Targeting Amyloid Intermediates by Design and Selection

描述(由申请人提供)： 阿尔茨海默病是一种毁灭性的神经系统疾病，其特征是患者大脑中淀粉样β-多肽(Ab)和神经原纤维缠结的细胞外沉积。遗传、病理和生化证据清楚地支持抗体聚集体和阿尔茨海默病(AD)进展之间的因果联系。最近的实验有力地表明，抗体的毒性可能存在于可溶性低聚中间体，也称为抗体衍生的可扩散配体(ADDL)。这些研究的预测是，无论是抑制中间体的形成还是破坏中间体的寡聚，都将对AD有治疗作用。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出新功能的β-折叠呈现蛋白，如抗体结合。我们的方法已经产生了一组具有预先组织的β-折叠表位的令人兴奋的蛋白质，这些蛋白质可以与抗体发生不同的相互作用，其中一些可溶性蛋白质强烈抑制抗体纤维的形成，而另一些蛋白质强烈促进抗体纤维的形成。在这项建议中，我们试图严格建立我们的淀粉样蛋白结合蛋白(称为HTB1变异体)的作用机制及其对抗体产生的细胞毒性的影响。我们建议(1)鉴定10个抗抗体的HTB1突变体改变抗体纤化率的能力，并直接测量它们与抗体纤维的结合；(2)利用化学交联、沉降速度和动态光散射来表征HTB1突变体与抗体纤化途径早期和后期中间体的结合；(3)表征HTB1突变体对抗体介导的细胞神经毒性的影响。最终目标是将这些实验中获得的各种物理、动力学和词汇学参数联系起来，以提出合理的治疗AD的策略。

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.