Lipoate Derivatives Targeting Alzheimer's Amyloid

描述（由申请人提供）：目前抗氧化/金属螯合治疗阿尔茨海默病（AD）的临床获益有限，因为它们的疾病靶标特异性较差。AD是最常见的老年痴呆症。此外，抗氧化/金属螯合作用对AD淀粉样蛋白病理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“药效团偶联”概念，设计靶向性抗氧化剂/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学机制。这一提议的具体假设是，淀粉样蛋白靶向抗氧化剂/金属螯合剂对大脑淀粉样蛋白病理和相关氧化应激具有靶向阻断作用。我们合成并表征了一种新的双功能抗氧化剂- XH2，它具有化学偶联淀粉样蛋白结合（苯并噻唑）和金属螯合抗氧化剂（脂酸盐）。我们的假设基于先前的观察和目前的试点数据表明：(1)生物金属，如铁、铜、锌，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，这是由活性氧（ROS）如H2O2介导的，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原子和1个O原子（<10个氢键受体），分子量=415(<500)，测得辛醇/水分配系数= 2.1¿0.1（<5）。它很符合利平斯基的五法则；（iii）计算得到XH2与A¿1-40肽相互作用，在其新制备的溶液中，对A¿1-40肽单体分子的亲和结合常数KD=4.43¿M；（iv） XH2在低微摩尔浓度下无神经毒性，且能减轻PS1/APP双转基因小鼠的脑A¿淀粉样蛋白病理，无明显动物毒性和行为障碍。我们计划通过以下三个具体目标来检验我们目前的假设，并实现本应用程序的目标：1。确定XH2对细胞培养中A¿神经毒性和APP翻译的影响2. 测定聚集和混合A¿1-40/42肽的XH2结合常数和XH2对血脑屏障（BBB）的渗透；3. 确定XH2对PS1/APP转基因小鼠脑A¿淀粉样蛋白病理、蛋白谷胱甘肽化和淀粉样斑块生物金属谱的影响。为了实现这些特定的目标，一系列的实验技术，如表面等离子体共振（SPR）， LC/MS, SDS-PAGE， ELISA，半定量免疫组织化学，激光捕获显微解剖（LCM）结合x射线荧光显微镜（微xrm）技术，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，将被用于提出的研究。该建议的总体原理是：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.