Testing different modifiers of the PERK pathway.

The activation of the unfolded protein response, particularly via the PERK pathway, has been suggested as a promising therapeutic approach in tauopathies, a group of neurodegenerative disorders characterized by the abnormal phosphorylation and aggregation of tau protein. So far, a shortage of available direct PERK activators has been limiting the progresses in this field. Our study aimed at the development of a cell-free screening assay enabling the detection of novel direct PERK activators. By applying the catalytic domain of recombinant human PERK, we initially determined ideal conditions of the kinase assay reaction, including parameters such as optimal kinase concentration, temperature, and reaction time. Instead of using PERK’s natural substrate proteins, eIF2α and NRF2, we applied SMAD3 as phosphorylation-accepting protein and successfully detected cell-free PERK activation and inhibition by selected modulators (e.g., calcineurin-B, GSK2606414). The developed assay revealed to be sufficiently stable and robust to assess an activating EC50-value. Additionally, our results suggested that PERK activation may take place independent of the active site which can be blocked by a kinase inhibitor. Finally, we confirmed the applicability of the assay by measuring PERK activation by MK-28, a recently described PERK activator. Overall, our data show that a cell-free luciferase-based assay with the recombinant human PERK kinase domain and SMAD3 as substrate protein is capable of detecting PERK activation, which enables to screen large compound libraries for direct PERK activators, in a high-throughput-based approach. These activators will be useful for deepening our understanding of the PERK signaling pathway, and may also lead to the identification of new therapeutic drug candidates for neurodegenerative tauopathies.蛋白激酶R样内质网激酶（PERK）通路介导的未折叠蛋白反应（unfolded protein response）激活，已被提出是tau蛋白病（tauopathies）——一类以tau蛋白异常磷酸化与聚集为特征的神经退行性疾病——中极具潜力的治疗策略。迄今为止，可用的直接PERK激活剂匮乏，掣肘了该领域的研究进展。本研究旨在开发一种无细胞筛选实验体系，以实现新型直接PERK激活剂的高效检测。通过采用重组人PERK的催化结构域，我们初步确定了激酶检测反应的最优条件，涵盖最优激酶浓度、反应温度及反应时长等参数。本研究未采用PERK的天然底物蛋白eIF2α与NRF2，而是选用SMAD3作为磷酸化受体蛋白，并成功通过选定的调节剂（如钙调神经磷酸酶-B、GSK2606414）检测到无细胞体系中的PERK激活与抑制效应。所构建的检测体系具备优异的稳定性与鲁棒性，可用于测定激活型半数有效浓度（EC50）值。此外，我们的研究结果表明，PERK激活可能独立于可被激酶抑制剂阻断的活性位点发生。最后，我们通过测定MK-28（一种新近报道的PERK激活剂）对PERK的激活作用，验证了该检测体系的适用性。总体而言，我们的研究数据表明，以重组人PERK激酶结构域与SMAD3作为底物蛋白的无细胞荧光素酶检测法，能够有效检测PERK的激活状态，从而可依托高通量手段筛选大型化合物库以获取直接PERK激活剂。此类激活剂将有助于加深我们对PERK信号通路的认知，亦有望为神经退行性tau蛋白病筛选出新的治疗候选药物。