Kalita Sujan, Kalita Sourav, Kawa Altaf Hussain, Shill Sukesh, Gupta Anjali, Kumar Sachin, Mandal Bhubaneswar
Laboratory of Peptide and Amyloid Research, Department of Chemistry, Indian Institute of Technology Guwahati Assam-781039 India
Department of Bioscience and Bioengineering, Indian Institute of Technology Guwahati Assam-781039 India.
RSC Med Chem. 2022 May 9;13(6):761-774. doi: 10.1039/d2md00019a. eCollection 2022 Jun 22.
Misfolding of the amyloid-β peptide (Aβ) and its subsequent aggregation into toxic oligomers is one of the leading causes of Alzheimer's disease (AD). As a therapeutic approach, cyclic peptides have been modified in many ways and developed as a potential class of amyloid aggregation inhibitors. Head-to-tail cyclic peptides with alternating d, l amino acids inhibit amyloid aggregation significantly. On the other hand, excess deposition of copper, iron, and zinc enhances amyloid aggregation. Dysregulation of these metal ions in the brain triggers aggregation by binding to the Aβ peptide. Therefore, specific metal chelators have been developed for disrupting the Aβ-metal complex, thereby reducing toxicity and restoring metal ion homeostasis. Herein, we report the development of a head-to-tail cyclic peptidomimetic with a copper chelating ligand attached. The designed peptidomimetic inhibits amyloid aggregation significantly in a two-fold molar ratio to the Aβ peptide, as confirmed by the thioflavin T (ThT) fluorescence assay, dynamic light scattering (DLS), transmission electron microscopy (TEM), and Congo-red stained birefringence studies. The chelating ligand attached to the cyclic peptide binds efficiently to Cu but weakly to Zn and Fe, thereby exhibiting profound selectivity, probed using UV-visible spectroscopy, thioflavin T (ThT) fluorescence assay, tyrosine (TYR10) fluorescence assay, isothermal titration calorimetry (ITC) and transmission electron microscopy (TEM). The non-toxicity of the designed peptidomimetics and their ability to reduce aggregating Aβ-fragment induced cytotoxicity was confirmed by the MTT assay on the mouse neuronal cell line. Further, the molecular interaction between the peptidomimetics and the Aβ-fragment was confirmed by Förster resonance energy transfer (FRET) studies using fluorescently labeled analogs. Cytotoxicity and cell internalization were also confirmed. A preliminary mechanistic investigation indicates that the peptidomimetic works by a synergistic effect of conformational restriction and metal sequestration. Such peptidomimetics can shed light on the mechanism of aggregation and a novel therapeutic approach.
淀粉样β肽（Aβ）的错误折叠及其随后聚集成有毒寡聚体是阿尔茨海默病（AD）的主要病因之一。作为一种治疗方法，环肽已在许多方面进行了修饰，并被开发为一类潜在的淀粉样蛋白聚集抑制剂。具有交替d、l氨基酸的头对尾环肽能显著抑制淀粉样蛋白聚集。另一方面，铜、铁和锌的过量沉积会增强淀粉样蛋白聚集。大脑中这些金属离子的失调通过与Aβ肽结合引发聚集。因此，已开发出特异性金属螯合剂来破坏Aβ-金属复合物，从而降低毒性并恢复金属离子稳态。在此，我们报告了一种连接有铜螯合配体的头对尾环肽模拟物的开发。通过硫黄素T（ThT）荧光测定、动态光散射（DLS）、透射电子显微镜（TEM）和刚果红染色双折射研究证实，所设计的肽模拟物以与Aβ肽2倍摩尔比显著抑制淀粉样蛋白聚集。连接到环肽上的螯合配体与铜有效结合，但与锌和铁结合较弱，从而表现出显著的选择性，这通过紫外可见光谱、硫黄素T（ThT）荧光测定、酪氨酸（TYR10）荧光测定、等温滴定量热法（ITC）和透射电子显微镜（TEM）进行了探究。通过对小鼠神经元细胞系的MTT测定证实了所设计肽模拟物的无毒性及其降低聚集性Aβ片段诱导的细胞毒性的能力。此外，使用荧光标记类似物的Förster共振能量转移（FRET）研究证实了肽模拟物与Aβ片段之间的分子相互作用。细胞毒性和细胞内化也得到了证实。初步的机制研究表明，肽模拟物通过构象限制和金属螯合的协同作用发挥作用。此类肽模拟物可为聚集机制和新型治疗方法提供线索。
