以Tau蛋白为靶点的抗阿尔茨海默症药物研发进展

阿尔茨海默病(Alzheimer's Disease,AD)是一种神经退行性疾病,主要发病特征为学习记忆下降和认知损伤.关于其发病机制主要有神经递质失调假说、β淀粉样蛋白假说、Tau蛋白假说以及慢性炎症假说等.目前,尚未有药物可以阻止和逆转AD的病情,本文主要综述了Tau蛋白假说的临床药物研发现状,为进一步研究开发靶向...     

喹啉类蛋白酪氨酸激酶抑制剂的研究进展

蛋白酪氨酸激酶的过度表达与多种肿瘤的发生、发展及转移相关,抑制酪氨酸激酶活性可有效抑制肿瘤。目前已有11个小分子蛋白酪氨酸激酶抑制剂作为抗肿瘤药物上市,1个由美国FDA批准接受预登记的药物,超过100个候选药物处于临床试验阶段。其中喹唑啉结构的酪氨酸激酶抑制剂已成为临床上最常见的抗肿瘤化疗药物类别之一。综述了近年来与喹唑啉结构相似的具有喹啉骨架的酪氨酸激酶抑制剂研究进展。     

小分子蛋白酪氨酸激酶抑制剂的研究进展

蛋白酪氨酸激酶是一类具有酪氨酸激酶活性的蛋白质,其异常表达与肿瘤的侵袭、转移以及肿瘤新生血管的生成等密切相关。以蛋白酪氨酸激酶作为靶点的药物研究已取得了突破性进展,目前已有二十余个小分子蛋白酪氨酸激酶抑制剂用于临床肿瘤的治疗,并有大量的抑制剂处于临床前和临床研究阶段。对近年来小分子蛋白酪氨酸激酶抑制剂的研究进展进行了综述。     

Coulter计数仪在尿结石形成动力学研究中的运用及其进展

通过检测粒子的尺寸和分布,库尔特(Coulter)颗粒计数仪可以研究泌尿系结石的生长和聚集动力学过程,比较防石药物的抑制指数。重点综述了在尿大分子、尿小分子,植物提取液和动物实验研究中Coulter计数仪的应用研究及其进展,并指出了其存在的不足。     

小分子伴侣对淀粉样蛋白β聚集抑制作用研究

淀粉样蛋白β(Aβ)的积累和聚集在阿尔茨海默症的致病机理研究中是非常关键的,因而开发能有效抑制Aβ聚集的新型抑制剂具有重要的意义。来源于大肠杆菌分子伴侣GroEL/GroES系统的小分子伴侣(GroEL顶端结构域191-345位残基)可以通过疏水相互作用与目标蛋白结合,具有生物相容性好等特点,是一种潜在的Aβ聚集抑制剂。通过硫代硫磺素T荧光光谱、透射电镜、凝胶过滤色谱分析和细胞毒性实验详细研究了小分子伴侣与Aβ_(42)的相互作用,并与姜黄素、表没食子儿茶素没食子酸酯和槲皮素等多酚类抑制剂的作用进行比较。结果表明小分子伴侣不仅降低了Aβ_(42)的聚集程度,同时也改变了Aβ_(42)的聚集路径,使得其聚集体分子量和纤维形貌发生了显著变化。相较于对照组,添加小分子伴侣后使得Aβ_(42)溶液原位ThT荧光响应值下降了72%,形成的纤维长度从数百纳米下降到小于50 nm,SH-SY5Y的细胞存活率提升了20%。因此,小分子伴侣能有效阻止Aβ纤维聚集体形成,降低其细胞毒性,具有良好的应用前景。     

基于亲脂性阳离子线粒体靶向化合物研究进展

亲脂性阳离子是一类能够通过脂质双分子层,并在肿瘤细胞高线粒体膜电位的驱动下,聚集于线粒体内的化合物。它们可与活性小分子化合物共价结合,从而作为载体,靶向传送活性化合物于肿瘤细胞线粒体中,实现对肿瘤的特异性治疗。该文将着重介绍离域化的亲脂性阳离子(DLC)介导的靶向小分子化合物研究,并对本课题组目前任务进行简要介绍。     

替卡格雷的合成研究进展

替卡格雷是一种新型选择性小分子抗凝血药物,能可逆性地作用于血管平滑肌细胞上的嘌呤2受体亚型P2Y12,明显抑制二磷酸腺苷(ADP)引起的血小板聚集。对替卡格雷的合成方法进行了归纳和比较,以期为进一步研究及工业化生产提供参考。     

具有生物活性杂环化合物的研究

开发新药剂的第一阶段是寻求先驱(提示)化合物。现在认为,以天然化合物为模式设计化合物及按研究对象的生理生化特性进行药物设计是行之有效的方法。本文就以天然化合物的化学与生物化学为依据,进行具有生物活性的新的杂环化合物之分子设计,由此对抗保幼激素、昆虫生长抑制、杀虫和植物生长抑制等化合物的研究情况作一介绍。     

抗肿瘤药物盐酸埃罗替尼的合成

盐酸埃罗替尼是一种4-苯胺基喹唑啉类的小分子化合物,主要以蛋白酪氨酸激酶为中心的一种抗肿瘤药物,对治疗非小细胞的肺癌、胰腺癌等有重要作用。针对抗肿瘤药物盐酸埃罗替尼的合成进行分析,对盐酸埃罗替尼合成工艺进行分析,旨在探讨研究一条高回报、低成本的商业化盐酸埃罗替尼合成道路。     

有机小分子催化合成胺类化合物的研究进展

在大部分药物分子和天然产物中,通常都含有胺类功能基,而通过有机小分子催化来直接构建C-N键,是合成胺类和酰胺类药物小分子结构的常用策略。该类化合物的传统合成方法,一般需要多种金属催化剂,甚至要用到危险化学试剂。有机小分子催化合成方法提供了一种制备有机胺类化合物且具有可持续和高经济性的合成路线,同时避免了金属污染。该文章旨在简述近年来有机小分子催化C-N键的形成,合成胺类化合物的新发现。     

Non-Canonical Roles of Tau and Their Contribution to Synaptic Dysfunction

Tau plays a central role in a group of neurodegenerative disorders collectively named tauopathies. Despite the wide range of diverse symptoms at the onset and during the progression of the pathology, all tauopathies share two common hallmarks, namely the misfolding and aggregation of Tau protein and progressive synaptic dysfunctions. Tau aggregation correlates with cognitive decline and behavioural impairment. The mechanistic link between Tau misfolding and the synaptic dysfunction is still unknown, but this correlation is well established in the human brain and also in tauopathy mouse models. At the onset of the pathology, Tau undergoes post-translational modifications (PTMs) inducing the detachment from the cytoskeleton and its release in the cytoplasm as a soluble monomer. In this condition, the physiological enrichment in the axon is definitely disrupted, resulting in Tau relocalization in the cell soma and in dendrites. Subsequently, Tau aggregates into toxic oligomers and amyloidogenic forms that disrupt synaptic homeostasis and function, resulting in neuronal degeneration. The involvement of Tau in synaptic transmission alteration in tauopathies has been extensively reviewed. Here, we will focus on non-canonical Tau functions mediating synapse dysfunction.     

Human Polymerase δ-Interacting Protein 2 (PolDIP2) Inhibits the Formation of Human Tau Oligomers and Fibrils

A central characteristic of Alzheimer’s disease (AD) and other tauopathies is the accumulation of aggregated and misfolded Tau deposits in the brain. Tau-targeting therapies for AD have been unsuccessful in patients to date. Here we show that human polymerase δ-interacting protein 2 (PolDIP2) interacts with Tau. With a set of complementary methods, including thioflavin-T-based aggregation kinetic assays, Tau oligomer-specific dot-blot analysis, and single oligomer/fibril analysis by atomic force microscopy, we demonstrate that PolDIP2 inhibits Tau aggregation and amyloid fibril growth in vitro. The identification of PolDIP2 as a potential regulator of cellular Tau aggregation should be considered for future Tau-targeting therapeutics.     

Proline-directed random-coil chemical shift values as a tool for the NMR assignment of the tau phosphorylation sites

NMR spectroscopy of the full-length neuronal Tau protein has proved to be difficult due to the length of the protein and the unfavorable amino acid composition. We show that the random-coil chemical shift values and their dependence on the presence of a proline residue in the (i+1) position can successfully be exploited to assign all proline-directed phosphorylation sites. This is a first step toward the study of the phosphorylation of Tau by NMR spectroscopy.     

Phosphorylation but Not Oligomerization Drives the Accumulation of Tau with Nucleoporin Nup98

Tau is a neuronal protein that stabilizes axonal microtubules (MTs) in the central nervous system. In Alzheimer’s disease (AD) and other tauopathies, phosphorylated Tau accumulates in intracellular aggregates, a pathological hallmark of these diseases. However, the chronological order of pathological changes in Tau prior to its cytosolic aggregation remains unresolved. These include its phosphorylation and detachment from MTs, mislocalization into the somatodendritic compartment, and oligomerization in the cytosol. Recently, we showed that Tau can interact with phenylalanine-glycine (FG)-rich nucleoporins (Nups), including Nup98, that form a diffusion barrier inside nuclear pore complexes (NPCs), leading to defects in nucleocytoplasmic transport. Here, we used surface plasmon resonance (SPR) and bio-layer interferometry (BLI) to investigate the molecular details of Tau:Nup98 interactions and determined how Tau phosphorylation and oligomerization impact the interactions. Importantly, phosphorylation, but not acetylation, strongly facilitates the accumulation of Tau with Nup98. Oligomerization, however, seems to inhibit Tau:Nup98 interactions, suggesting that Tau-FG Nup interactions occur prior to oligomerization. Overall, these results provide fundamental insights into the molecular mechanisms of Tau-FG Nup interactions within NPCs, which might explain how stress-and disease-associated posttranslational modifications (PTMs) may lead to Tau-induced nucleocytoplasmic transport (NCT) failure. Intervention strategies that could rescue Tau-induced NCT failure in AD and tauopathies will be further discussed.     

重组人Tau蛋白的表达、纯化及鉴定

目的 重组人Tau蛋白的诱导表达和纯化。方法 将携带 Tau蛋白基因的工程菌株经IPTG诱导表达后,通过超声破碎、硫酸按盐析、离子交换层析、电洗脱等方法纯化Tau蛋白,以SDS-PAGE、非变性PAGE、Westem blot和N末端氨基酸测定等方法进行鉴定。结果 纯化Tau蛋白的得率为28.5％,在SDS-PAGE和非变性PAGE上呈均一的蛋白条带,其相对分子质量约为59000,N末端5个氨基酸测定结果为NH2-Met-Ala-Glu-Pro-Arg-。结论 本实验采用纯化重组人Tau蛋白方法是可行的。     

Deciphering the Effect of Lysine Acetylation on the Misfolding and Aggregation of Human Tau Fragment 171IPAKTPPAPK180 Using Molecular Dynamic Simulation and the Markov State Model

The formation of neurofibrillary tangles (NFT) with β-sheet-rich structure caused by abnormal aggregation of misfolded microtubule-associated protein Tau is a hallmark of tauopathies, including Alzheimer’s Disease. It has been reported that acetylation, especially K174 located in the proline-rich region, can largely promote Tau aggregation. So far, the mechanism of the abnormal acetylation of Tau that affects its misfolding and aggregation is still unclear. Therefore, revealing the effect of acetylation on Tau aggregation could help elucidate the pathogenic mechanism of tauopathies. In this study, molecular dynamics simulation combined with multiple computational analytical methods were performed to reveal the effect of K174 acetylation on the spontaneous aggregation of Tau peptide ¹⁷¹IPAKTPPAPK¹⁸⁰, and the dimerization mechanism as an early stage of the spontaneous aggregation was further specifically analyzed by Markov state model (MSM) analysis. The results showed that both the actual acetylation and the mutation mimicking the acetylated state at K174 induced the aggregation of the studied Tau fragment; however, the effect of actual acetylation on the aggregation was more pronounced. In addition, acetylated K174 plays a major contributing role in forming and stabilizing the antiparallel β-sheet dimer by forming several hydrogen bonds and side chain van der Waals interactions with residues I171, P172, A173 and T175 of the corresponding chain. In brief, this study uncovered the underlying mechanism of Tau peptide aggregation in response to the lysine K174 acetylation, which can deepen our understanding on the pathogenesis of tauopathies.     

Accepting its random coil nature allows a partial NMR assignment of the neuronal Tau protein

A combined strategy to obtain a partial NMR assignment of the neuronal Tau protein is presented. Confronted with the extreme spectral degeneracy that the spectrum of this 441 amino acid long unstructured protein presents, we have introduced a graphical procedure based on residue type-specific product planes. Combining this strategy with the search for pairwise motifs, and combining the spectra of different Tau isoforms and even of peptides derived from the native sequence, we arrive at a partial assignment that is sufficient to map the interactions of Tau with its molecular partners. The obtained assignments equally confirm the absence of regular secondary structure in the isolated protein.     

Potent Tau Aggregation Inhibitor D-Peptides Selected against Tau-Repeat 2 Using Mirror Image Phage Display

Alzheimer's disease and other Tauopathies are associated with neurofibrillary tangles composed of Tau protein, as well as toxic Tau oligomers. Therefore, inhibitors of pathological Tau aggregation are potentially useful candidates for future therapies targeting Tauopathies. Two hexapeptides within Tau, designated PHF6* (275-VQIINK-280) and PHF6 (306-VQIVYK-311), are known to promote Tau aggregation. Recently, the PHF6* segment has been described as the more potent driver of Tau aggregation. We therefore employed mirror-image phage display with a large peptide library to identify PHF6* fibril binding peptides consisting of D-enantiomeric amino acids. The suitability of D-enantiomeric peptides for in vivo applications, which are protease stable and less immunogenic than L-peptides, has already been demonstrated. The identified D-enantiomeric peptide MMD3 and its retro-inverso form, designated MMD3rev, inhibited in vitro fibrillization of the PHF6* peptide, the repeat domain of Tau as well as full-length Tau. Dynamic light scattering, pelleting assays and atomic force microscopy demonstrated that MMD3 prevents the formation of tau β-sheet-rich fibrils by diverting Tau into large amorphous aggregates. NMR data suggest that the D-enantiomeric peptides bound to Tau monomers with rather low affinity, but ELISA (enzyme-linked immunosorbent assay) data demonstrated binding to PHF6* and full length Tau fibrils. In addition, molecular insight into the binding mode of MMD3 to PHF6* fibrils were gained by in silico modelling. The identified PHF6*-targeting peptides were able to penetrate cells. The study establishes PHF6* fibril binding peptides consisting of D-enantiomeric amino acids as potential molecules for therapeutic and diagnostic applications in AD research.     

Zebrafish Models to Study New Pathways in Tauopathies

Tauopathies represent a vast family of neurodegenerative diseases, the most well-known of which is Alzheimer’s disease. The symptoms observed in patients include cognitive deficits and locomotor problems and can lead ultimately to dementia. The common point found in all these pathologies is the accumulation in neural and/or glial cells of abnormal forms of Tau protein, leading to its aggregation and neurofibrillary tangles. Zebrafish transgenic models have been generated with different overexpression strategies of human Tau protein. These transgenic lines have made it possible to highlight Tau interacting factors or factors which may limit the neurotoxicity induced by mutations and hyperphosphorylation of the Tau protein in neurons. Several studies have tested neuroprotective pharmacological approaches. On few-days-old larvae, modulation of various signaling or degradation pathways reversed the deleterious effects of Tau mutations, mainly hTauP301L and hTauA152T. Live imaging and live tracking techniques as well as behavioral follow-up enable the analysis of the wide range of Tau-related phenotypes from synaptic loss to cognitive functional consequences.