Shaik Jeelan Basha, Kandrakonda Yelamanda Rao, Kallubai Monika, Gajula Navya Naidu, Dubey Shreya, Aramati Bindu Madhava Reddy, Subramanyam Rajagopal, Amooru Gangaiah Damu
Department of Chemistry, Yogi Vemana University, Kadapa, Andhra Pradesh, India.
Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India.
Int J Biol Macromol. 2021 Dec 15;193(Pt B):1409-1420. doi: 10.1016/j.ijbiomac.2021.10.204. Epub 2021 Nov 2.
Investigating the drug-AChE binding mechanism is vital in understanding its cogent use in medical practice against Alzheimer's disease (AD). The production and accumulation of oligomers of β-amyloid is a central event in the neuropathology of AD. Beside the inhibition of assembly process, modulation of the aggregation process of these proteins towards minimally toxic pathways may be a possible therapeutic strategy for AD. Hence, the present study aims to examine the effect of multifunctional fused tricyclic 7-hydroxy 4-methyl coumarin analogs (HMC) on the self-induced aggregation of β-amyloid using Thioflavin T (ThT) assay, scanning electron microscopic study, AlamarBlue and immune blotting assays and also the binding mechanism with AChE by fluorescence emission, conformational, molecular docking and molecular dynamic simulation studies under physiological pH 7.4. The ThT assay, FE-SEM study, cell line and western blots establish that the HMC molecules could irreversibly disrupt preformed Aβ fibrils, accelerate the aggregates into micro size co-assembled structures, and effectively eliminate the cytotoxicity of Aβ. Fluorescence emission studies indicating a strong binding affinity between HMC and AChE with the binding constants of 1.04 × 10, 3.57 × 10, 1.97 × 10, 3.07 × 10 and 2.95 × 10 M, respectively and binding sites number found to be 1. CD studies disclosed a partial unfolding in the secondary structure of AChE upon binding with HMC. Docking analysis inferred that the HMC were bound through hydrophobic and hydrophilic interactions to the AChE active site. Molecular dynamics simulations emphasized the stability of AChE-HMC complexes throughout the 100 ns simulations, and the local conformational changes of the residues of AChE validate the stability of complexes. These results provide new and unique complementary approach for modulating the biological effects of the Aβ aggregates by coumarin analogs and new insights for further in vivo investigations as novel anti AD agents.
研究药物与乙酰胆碱酯酶（AChE）的结合机制对于理解其在医学实践中有效治疗阿尔茨海默病（AD）至关重要。β-淀粉样蛋白寡聚体的产生和积累是AD神经病理学中的核心事件。除了抑制组装过程外，将这些蛋白质的聚集过程调节至毒性最小的途径可能是治疗AD的一种可行策略。因此，本研究旨在使用硫黄素T（ThT）测定法、扫描电子显微镜研究、AlamarBlue和免疫印迹测定法，研究多功能稠合三环7-羟基-4-甲基香豆素类似物（HMC）对β-淀粉样蛋白自诱导聚集的影响，以及在生理pH 7.4条件下通过荧光发射、构象、分子对接和分子动力学模拟研究其与AChE的结合机制。ThT测定法、场发射扫描电子显微镜（FE-SEM）研究、细胞系和蛋白质印迹法证实，HMC分子可以不可逆地破坏预先形成的Aβ纤维，加速聚集体形成微米级共组装结构，并有效消除Aβ的细胞毒性。荧光发射研究表明HMC与AChE之间具有很强的结合亲和力，结合常数分别为1.04×10、3.57×10、1.97×10、3.07×10和2.95×10 M，且结合位点数为1。圆二色性（CD）研究表明，AChE与HMC结合后二级结构发生部分解折叠。对接分析推断，HMC通过疏水和亲水相互作用与AChE活性位点结合。分子动力学模拟强调了在整个100 ns模拟过程中AChE-HMC复合物的稳定性，AChE残基的局部构象变化验证了复合物的稳定性。这些结果为香豆素类似物调节Aβ聚集体的生物学效应提供了新的独特互补方法，并为作为新型抗AD药物的进一步体内研究提供了新的见解。
