Yu Shao Feng, Michon Maya, Lingappa Anuradha F, Paulvannan Kumar, Solas Dennis, Staats Kim, Ichida Justin, Dey Debendranath, Rosenfeld Jeffrey, Lingappa Vishwanath R
Prosetta Biosciences, Inc., 670 5th St, San Francisco, CA, 94107, USA.
University of Southern California, Los Angeles, CA, 90033, USA.
Clin Proteomics. 2025 Apr 28;22(1):16. doi: 10.1186/s12014-025-09538-4.
Assembly modulators are a new class of allosteric site-targeted therapeutic small molecules, some of which are effective at restoring nuclear localization of TDP-43 in ALS cellular models, and which display efficacy in a variety of ALS animal models. These compounds have been shown to bind selectively to a small subset of protein disulfide isomerase (PDI), a protein implicated in ALS pathophysiology. The targeted subset of PDI is found within a novel, transient and energy-dependent multi-protein complex that includes other important members of the ALS interactome, such as TDP-43, RanGTPase, and selective autophagy receptor p62/SQSTM1. We demonstrate here that a similar multi-protein complex drug target is present in PBMCs as isolated by energy-dependent drug resin affinity chromatography (eDRAC) and characterized by mass spectrometry and by Western blot (WB). Signature alterations in the composition of the multi-protein complex in PBMCs from ALS patients compared to PBMCs from healthy individuals were identified by WB of eDRAC bound proteins, thereby extending earlier literature suggesting PBMC dysfunction in ALS. Changes in the PBMC drug target in ALS patients compared to healthy individuals include diminished p62/SQSTM1 and appearance of a 17 kDa post-translationally modified form of RanGTPase. These changes are not readily apparent from analysis of whole cell extracts, as the individual protein components within the drug target multi-protein complex comprise only small percentages of the total of those component proteins in the extract. Furthermore, whole blood from ALS patients shows a distinctive degradation of total RanGTPase not observed in blood from healthy individuals. This degradation appears to be rescued by treatment of whole blood from ALS patients for 72 h with ALS-active assembly modulator small molecules. Our findings are consistent with the hypothesis that ALS is fundamentally a disorder of homeostasis that can be detected early, prior to disability, in blood by the methods described, and restored to the healthy state by assembly modulator drug treatment.
组装调节剂是一类新型的靶向变构位点的治疗性小分子，其中一些在肌萎缩侧索硬化症（ALS）细胞模型中能有效恢复TDP - 43的核定位，并且在多种ALS动物模型中显示出疗效。这些化合物已被证明能选择性结合一小部分蛋白质二硫键异构酶（PDI），该蛋白质与ALS病理生理学有关。PDI的靶向亚群存在于一种新型的、短暂的且依赖能量的多蛋白复合物中，该复合物包含ALS相互作用组的其他重要成员，如TDP - 43、RanGTP酶和选择性自噬受体p62/SQSTM1。我们在此证明，通过能量依赖型药物树脂亲和色谱法（eDRAC）分离并经质谱和蛋白质印迹法（WB）表征后，外周血单核细胞（PBMC）中存在类似的多蛋白复合物药物靶点。通过对eDRAC结合蛋白进行WB分析，确定了ALS患者的PBMC与健康个体的PBMC相比，多蛋白复合物组成的特征性改变，从而扩展了早期文献中关于ALS患者PBMC功能障碍的观点。与健康个体相比，ALS患者PBMC药物靶点的变化包括p62/SQSTM1减少以及出现一种17 kDa翻译后修饰形式的RanGTP酶。这些变化在全细胞提取物分析中并不明显，因为药物靶点多蛋白复合物中的单个蛋白质成分仅占提取物中这些成分总蛋白的小百分比。此外，ALS患者的全血显示出总RanGTP酶有明显降解，而健康个体的血液中未观察到这种降解。用具有ALS活性的组装调节剂小分子对ALS患者的全血进行72小时处理后，这种降解似乎得到了挽救。我们的研究结果与以下假设一致：ALS从根本上说是一种内稳态紊乱，通过所述方法在血液中可以在残疾之前早期检测到，并通过组装调节剂药物治疗恢复到健康状态。
