Somavarapu Arun Kumar, Kleijwegt Giulia, Nagaraj Madhu, Alam Parvez, Nielsen Janni, Otzen Daniel E
Interdisciplinary Nanoscience Center (iNANO), Aarhus University Gustav Wieds Vej 14, 8000 Aarhus C Denmark
Chem Sci. 2023 Feb 21;14(11):3030-3047. doi: 10.1039/d2sc05534a. eCollection 2023 Mar 15.
Small soluble oligomers of the protein α-synuclein (αSO) have been linked to disruptions in neuronal homeostasis, contributing to the development of Parkinson's Disease (PD). While this makes αSO an obvious drug target, the development of effective therapeutics against αSO is challenged by its low abundance and structural and morphological complexity. Here, we employ two different approaches to neutralize toxic interactions made by αSOs with different cellular components. First, we use available data to identify four neuronal proteins as likely candidates for αSO interactions, namely Cfl1, Uchl1, Sirt2 and SerRS. However, despite promising results when immobilized, all 4 proteins only bind weakly to αSO in solution in microfluidic assays, making them inappropriate for screening. In contrast, the formation of stable contacts formed between αSO and vesicles consisting of anionic lipids not only mimics a likely biological role of αSO but also provided a platform to screen two small molecule libraries for disruptors of these contacts. Of the 7 best leads obtained in this way, 2 significantly impaired αSO contacts with other proteins in a sandwich ELISA assay using αSO-binding monoclonal antibodies and nanobodies. In addition, 5 of these leads suppressed α-synuclein amyloid formation. Thus, a repurposing screening that directly targets a key culprit in PD pathogenesis shows therapeutic potential.
蛋白质α-突触核蛋白(αSO)的小可溶性寡聚体与神经元内环境稳态的破坏有关,这促进了帕金森病(PD)的发展。虽然这使得αSO成为一个明显的药物靶点,但针对αSO开发有效的治疗方法受到其低丰度以及结构和形态复杂性的挑战。在这里,我们采用两种不同的方法来中和αSO与不同细胞成分产生的毒性相互作用。首先,我们利用现有数据确定四种神经元蛋白可能是αSO相互作用的候选蛋白,即Cfl1、Uchl1、Sirt2和SerRS。然而,尽管固定化时结果很有前景,但在微流控分析中,所有这4种蛋白在溶液中与αSO的结合都很弱,这使得它们不适合用于筛选。相比之下,αSO与由阴离子脂质组成的囊泡之间形成的稳定接触不仅模拟了αSO可能的生物学作用,还提供了一个平台来筛选两个小分子文库以寻找这些接触的破坏剂。通过这种方式获得的7个最佳先导化合物中,有2个在使用αSO结合单克隆抗体和纳米抗体的夹心ELISA分析中显著削弱了αSO与其他蛋白的接触。此外,这些先导化合物中有5个抑制了α-突触核蛋白淀粉样蛋白的形成。因此,直接针对PD发病机制中的关键罪魁祸首进行的重新利用筛选显示出治疗潜力。