Lipoate Derivatives Targeting Alzheimer's Amyloid

描述（由申请人提供）：目前针对阿尔茨海默病（AD）（最常见的老年痴呆症）的抗氧化剂/金属螯合疗法的临床益处由于其疾病靶标特异性较差而受到限制。此外，抗氧化剂/金属螯合作用对 AD 淀粉样蛋白病理学的确切作用机制尚不清楚。因此，我们的长期目标是基于新兴的药物开发“药效团缀合”概念，设计目标特异性抗氧化剂/金属螯合剂，并更好地了解其治疗 AD 和其他人类疾病的药理机制。该提议的具体假设是，针对淀粉样蛋白的抗氧化剂/金属螯合剂假设有针对性地阻止大脑淀粉样蛋白病理学和相关的氧化应激。我们合成并表征了一种新型双功能抗氧化剂 - XH2，其具有化学缀合的淀粉样蛋白结合（苯并噻唑）和金属螯合抗氧化剂（硫辛酸）部分。我们的假设基于之前的观察和当前的试点数据，表明：（i）生物金属（例如 Fe、Cu、Zn）强烈存在于人类淀粉样斑块中，促进 A¿ 淀粉样变性和寡聚化，这些淀粉样变性和寡聚化由活性氧（ROS）（例如 H2O2）介导，并被过氧化氢酶和金属螯合剂 - DTPA 和 EDTA 减弱； (ii) XH2分子只有1个NH基团（<5个氢键供体）、2个N和1个O原子（<10个氢键受体），MW=415（<500），测得的辛醇/水分配系数=2.1¿0.1（<5）。它与利平斯基一般的五法则非常吻合。 (iii) XH2 通过计算与 A¿1-40 肽相互作用，并且在其新鲜制备的溶液中对单体 A¿1-40 肽分子具有 KD = 4.43 µM 的亲和结合常数； (iv) XH2在低微摩尔浓度下没有神经毒性，并且它减弱PS1/APP双转基因小鼠的脑A¿淀粉样蛋白病理学，而不诱导明显的动物毒性和行为障碍。我们计划通过追求以下三个具体目标来检验我们当前的假设并实现本应用的目标： 1. 确定 XH2 对细胞培养中 A¿ 神经毒性和 APP 翻译的影响； 2. 确定聚集和混合的 A¿1-40/42 肽的 XH2 结合常数以及 XH2 的血脑屏障 (BBB) 穿透力； 3. 确定 XH2 对 PS1/APP 转基因小鼠的脑 A 淀粉样蛋白病理学、蛋白质谷胱甘肽化和淀粉样斑块中生物金属谱的影响。为了实现这些具体目标，一系列实验技术，如表面等离子共振（SPR）、LC/MS、SDS-PAGE、ELISA、半定量免疫组织化学、激光捕获显微切割（LCM）与X射线荧光显微镜（micro-XRM）技术相结合，以及体外（人SH-SY5Y神经母细胞瘤和E17原代大鼠皮质细胞）和体内（PS1/APP双转基因小鼠）AD模型，将用于AD模型拟议的研究。该提案的总体理由是：我们对淀粉样蛋白靶向抗氧化剂/螯合剂作为潜在 AD 修饰剂的分子机制和治疗价值的理解需要进一步澄清和验证。

DESCRIPTION (provided by applicant): The clinical benefits of current antioxidant/metal chelation therapies for Alzheimer's disease (AD), the most common form of senile dementia, are limited due to their poor disease target specificity. Moreover, the exact subserving mechanism(s) of antioxidant/metal chelation effects upon AD amyloid pathology are not clear. Our long-term objective is thus to design target-specific antioxidants/metal chelators and better understand their pharmacological mechanisms for AD and other human diseases, based on an emerging "pharmacophore conjugation" concept for drug development. The specific hypothesis for this proposal is that amyloid-targeting antioxidants/metal chelators assume targeted interdictions against cerebral A¿ amyloid pathology and associated oxidative stress. We have synthesized and characterized a novel bifunctional antioxidant- XH2 with chemically conjugated amyloid-binding (benzothiazole) and metal-chelating antioxidant (lipoate) moieties. We base the hypothesis on previous observations and current pilot data suggest that: (i) biometals such as Fe, Cu, Zn, which are strongly present in human amyloid plaques, promote A¿ amyloidosis and oligomerization that are mediated by reactive oxygen species (ROS) such as H2O2 and attenuated by catalase and metal chelators- DTPA and EDTA; (ii) XH2 molecule has only one NH group (<5 hydrogen-bond donors), two N and one O atoms (<10 hydrogen bond acceptors), MW=415 (<500), and the measured octanol/water partition coefficient = 2.1¿0.1 (<5). It agrees well with the general Lipinski's Rule of Five; (iii) XH2 interacts with A¿1-40 peptide computationally and has an affinity binding constant of KD=4.43 ¿M toward monomeric A¿1-40 peptide molecules in its freshly prepared solution; (iv) XH2 has no neurotoxicity at low micromolar concentrations, and it attenuates cerebral A¿ amyloid pathology in PS1/APP doubly transgenic mice without inducing apparent animal toxicity and behavior disturbances. We plan to test our current hypothesis and to achieve the objective of this application by pursuing the following three specific aims: 1. Determine the effects of XH2 upon A¿ neurotoxicity and APP translation in cell culture; 2. Determine XH2 binding constants for aggregated and mixed A¿1-40/42 peptides and blood-brain barrier (BBB) penetration of XH2; 3. Determine the effects of XH2 upon cerebral A¿ amyloid pathology, protein glutathionylation, and biometal profiles in amyloid plaques from PS1/APP transgenic mice. To achieve these specific aims, an array of experimental techniques such as surface plasmon resonance (SPR), LC/MS, SDS-PAGE, ELISA, semi-quantitative immunohistochemistry, laser capture microdissection (LCM) coupled with x-ray fluorescence microscopy (micro-XRM) techniques, and in vitro (human SH-SY5Y neuroblastoma and E17 primary rat cortical cells) and in vivo (PS1/APP doubly transgenic mouse) AD models, will be employed for the proposed studies. The overall rationale for the proposal is: our understanding about the molecular mechanism(s) and therapeutic values of amyloid-targeted antioxidants/chelators as potential AD-modifying agents need to be further clarified and validated.