Targeting Amyloid Intermediates by Design and Selection

描述（由申请人提供）： 阿尔茨海默病是一种破坏性的神经系统疾病，其特征在于患者脑中淀粉样β-肽（Ab）的细胞外沉积和神经元缠结。遗传学、病理学和生物化学证据清楚地支持Ab聚集体与阿尔茨海默病（AD）进展之间的因果关系。最近的实验强烈表明，抗体的毒性可能在于可溶性寡聚体中间体，也称为抗体衍生的扩散配体（ADDL）。这些研究的预测是，抑制中间体的形成或破坏中间体的寡聚化将在AD中具有治疗益处。我们最近建立了一种新的双重选择策略，依赖于噬菌体展示，选择保留其原始结构并进化新功能（如Ab结合）的β-折叠呈递蛋白。我们的方法已经产生了一组令人兴奋的蛋白质与预组织的β-折叠表位，可以与抗体，其中一些可溶性蛋白质强烈抑制抗体原纤维形成，而其他蛋白质强烈加速抗体原纤维形成的差异相互作用。在该提案中，我们试图严格建立我们的淀粉样蛋白结合蛋白（称为hTB 1变体）的作用机制及其对Ab衍生的细胞毒性的影响。我们建议（1）鉴定10种针对Ab选择的hTB 1变体改变Ab纤维化速率的能力，并直接测量它们与Ab原纤维的结合;（2）利用化学交联表征hTB 1变体与沿着Ab固定化途径的早期和晚期阶段的中间体的结合，沉降速度和动态光散射，最后（3）表征hTB 1变体对Ab介导的细胞神经毒性的影响。最终目的是将这些实验中获得的各种物理、动力学和词汇学参数关联起来，以提出治疗AD的合理策略。

DESCRIPTION (provided by applicant): Alzheimer's disease is a devastating neurological disease characterized by extracellular deposits of amyloidbeta- peptides(Ab) and neurofibrillary tangles in the brains of patients. Genetic, pathological and biochemical evidence clearly support a causal link between Ab aggregates and Alzheimer's disease (AD) progression. Recent experiments strongly suggest that the toxicity of Ab may lie in the soluble oligomeric intermediates also called Ab-derived diffusible ligands (ADDL). The prediction of these studies is that either inhibiting the formation or disrupting the oligomerization of intermediates would be of therapeutic benefit in AD. We have recently established a novel dual selection strategy relying on phage-display that selects for beta-sheet presenting proteins that retain their original structure and evolve new functions, such as Ab binding. Our method has yielded an exciting set of proteins with preorganized beta-sheet epitopes that can interact differentially with Ab, where some soluble proteins strongly inhibit Ab fibril formation whereas other proteins strongly accelerate Ab fibril formation. In this proposal we seek to rigorously establish the mechanism of action of our amyloid-binding proteins (called hTB1 variants) and their effect upon Ab derived cellular toxicity. We propose to (1) characterize ten hTB1 variants selected against Ab for their ability to alter Ab fibrillization rates and also directly measure their binding to Ab fibrils; (2) characterize the binding of the hTB1 variants to intermediates along early and late stages of the Ab fibrillization pathway utlizing chemical crosslinking, sedimentation velocity and dynamic light scattering and finally (3) characterize the effect of the hTB1 variants upon the cellular neurotoxicity mediated by Ab. The ultimate objective is to correlate the various physical, kinetic and lexicological parameters obtained in these experiments to propose rational strategies for treating AD.