The purpose of our study is to understand the protective effects of small molecule ligands for phosphorylated tau (p-tau) in Alzheimer’s disease (AD) progression. Many reports show evidence that phosphorylated tau is reported to be an important contributor to the formation of paired helical filaments (PHFs) and neurofibrillary tangles (NFTs) in AD neurons. In AD, glycogen synthase kinase-3 beta (GSK3β), cyclin-dependent kinase-5 and dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), are the three important kinases responsible for tau hyperphosphorylation. Currently, there are no drugs and/or small molecules that reduce the toxicity of phosphorylated tau in AD. In the present study, we rationally selected and validated small molecule ligands that bind to the phosphorylated tau at SER23 (Ser 285) and TYR44 (Tyr310). We also assessed the molecular dynamics and validated molecular docking sites for the three best ligands. Based on the best docking scores −8.09, −7.9 and −7.8 kcal/mol, we found that ligand 1 binds to key hyperphosphorylation residues of phosphorylated tau that inhibit abnormal PHF-tau, DYRK1A and GKS3β that reduce phosphorylated tau levels in AD. Using biochemical, molecular, immunoblotting, immunofluorescence and transmission electron microscopy analyses, we studied the ligand 1 inhibition as well as mitochondrial and synaptic protective effects in immortalized primary hippocampal neuronal (HT22) cells. We found interactions between NAT10-262501 (ligand 1) and phosphorylated tau at key phosphorylation sites and these ligand-based inhibitions decreased PHF-tau, DYRK1A and GSK3β levels. We also found increased mitochondrial biogenesis, mitochondrial fusion and synaptic activities and reduced mitochondrial fission in ligand 1-treated mutant tau HT22 cells. Based on these results, we cautiously conclude that phosphorylated tau NAT10-262501 (ligand 1) reduces hyperphosphorylation of tau based GKS3β and CDK5 kinase regulation in AD, and aids in the maintenance of neuronal structure, mitochondrial dynamics and biogenesis with a possible therapeutic drug target for AD.

中文翻译：

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小分子抑制剂配体对阿尔茨海默病过度磷酸化 tau 诱导的线粒体和突触毒性的保护作用

我们研究的目的是了解小分子配体对磷酸化 tau (p-tau) 在阿尔茨海默病 (AD) 进展中的保护作用。许多报告显示，有证据表明磷酸化的 tau 据报道是 AD 神经元中成对螺旋丝 (PHF) 和神经原纤维缠结 (NFT) 形成的重要贡献者。在 AD 中，糖原合酶激酶 3 β (GSK3β)、细胞周期蛋白依赖性激酶 5 和双特异性酪氨酸磷酸化调节激酶 1A (DYRK1A) 是负责 tau 过度磷酸化的三种重要激酶。目前，没有药物和/或小分子可以降低 AD 中磷酸化 tau 的毒性。在本研究中，我们合理选择并验证了与 SER23 (Ser 285) 和 TYR44 (Tyr310) 磷酸化 tau 结合的小分子配体。我们还评估了三种最佳配体的分子动力学和验证的分子对接位点。基于最佳对接分数 -8.09、-7.9 和 -7.8 kcal/mol，我们发现配体 1 与磷酸化 tau 的关键过度磷酸化残基结合，抑制异常 PHF-tau、DYRK1A 和 GKS3β，从而降低 AD 中的磷酸化 tau 水平。使用生化、分子、免疫印迹、免疫荧光和透射电子显微镜分析，我们研究了永生化原代海马神经元 (HT22) 细胞中的配体 1 抑制以及线粒体和突触保护作用。我们发现 NAT10-262501（配体 1）和磷酸化 tau 在关键磷酸化位点之间的相互作用，这些基于配体的抑制降低了 PHF-tau、DYRK1A 和 GSK3β 水平。我们还发现线粒体生物合成增加，配体 1 处理的突变 tau HT22 细胞中的线粒体融合和突触活动并减少线粒体裂变。基于这些结果，我们谨慎地得出结论，磷酸化 tau NAT10-262501（配体 1）可降低 AD 中基于 tau 的 GKS3β 和 CDK5 激酶调节的过度磷酸化，并有助于维持神经元结构、线粒体动力学和生物发生，并具有可能的治疗药物靶点对于广告。