Targeting A-beta with evolved site-specific proteases

描述(由申请人提供)：在这项提案中，我们计划开发治疗性蛋白酶制剂，旨在针对阿尔茨海默病(AD)假定的潜在致病机制--淀粉样β蛋白(Ab)肽的积累--这被证明是小分子策略的困难靶点。具体地说，我们希望开发一种基于选择的定向进化方案来快速鉴定具有高选择性和对抗体中特定肽片段的催化活性的蛋白酶。最近的证据表明，淀粉样蛋白纤维和细胞毒性可溶性聚集体的形成是一个平衡控制的过程，由抗体的积累触发。此外，抗体的生物发生和清除机制的缺陷与AD的致病性有关。因此，我们假设，我们的催化剂将能够通过表现出针对淀粉样蛋白原肽的内部可扩增的活性来挽救抗体清除途径。具体地说，我们预计单抗的切割将能够1)将聚集平衡向较不复杂的物种移动，2)以蛋白分解产物的形式在原位产生有效的聚集抑制物。因此，我们相信，抗体的位置选择性蛋白分解为开发针对这种衰弱疾病的高效治疗策略提供了一个独特的机会，我们计划在开发这种方法时实现以下具体目标： 具体目标1：我们计划开发一种基因选择系统，以特定位点的蛋白酶作为起始支架，能够从大量遗传编码的序列排列中识别具有非本地抗抗体活性的独特活性物种。 具体目标2：我们将使用真正的淀粉样变性底物来确认进化的蛋白酶的抗抗体活性，并进一步表征这些制剂，使用1)底物专一性分析的动力学分析，2)评估它们的解聚特性的双相试验，以及3)测量它们的细胞保护倾向的细胞存活率试验。 具体目标3：最后，我们将通过提高抗抗体蛋白水解酶对系统代谢的稳定性和降低其非单一性来优化其药代动力学性质。 这项研究的预期结果将是鉴定能够通过催化将抗体单体降解为非纤维蛋白原性片段以及潜在的抗淀粉样蛋白片段来攻击可能的AD病原体的治疗性蛋白水解剂。我们的长期目标是开发治疗策略，针对AD的潜在致病机制。

DESCRIPTION (provided by applicant): In this proposal we plan to develop therapeutic protease agents designed to target the presumed underlying pathogenic mechanism in Alzheimer's disease (AD) - accumulation of amyloid-beta (Ab) peptide - which proved to be a difficult target for small-molecule strategies. Specifically, we wish to exploit a selection-based directed evolution scheme for rapid identification of proteases with high selectivities and catalytic activities against a specific peptide fragment within Ab. Recent evidence suggests that the formation of amyloid fibrils, as well as the cytotoxic soluble aggregates is an equilibrium-controlled process, triggered by accumulation of Ab. Moreover, defects in both biogenesis and clearance mechanisms of Ab have been linked to the pathogenicity of AD. We hypothesize, therefore, that our catalytic agents will be able to rescue the Ab clearance pathway by exhibiting an internally amplifiable activity against the amyloidogenic peptide. Specifically, we expect that the cleavage of monomoric Ab will be able to 1) shift the aggregation equilibrium toward less complex species and 2) generate potent aggregation inhibitors in situ in the form of proteolysis products. We believe, therefore, that the site-selective proteolysis of Ab presents a unique opportunity for the development of a highly effective therapeutic strategy against this debilitating disease and we plan to accomplish the following Specific Aims in developing this approach: Specific Aim 1: Using a site-specific protease as a starting scaffold, we plan to develop a genetic selection system capable of identifying uniquely active species with nonnative activities against Ab from a vast collection of genetically encoded sequence permutations. Specific Aim 2: We will confirm anti-Ab activities of the evolved proteases using an authentic amyloidogenic substrate and further characterize these agents, using 1) kinetic analysis for the substrate specificity analysis, 2) biphasic assays for assessment of their deaggregation properties, and 3) cell viability assay for the measure of their cytoprotective propensity. Specific Aim 3: Finally, we will optimize the pharmacokinetic properties of the anti-Ab proteases by improving their stability to systemic metabolism and reducing their immonogenicity. The expected outcome of this research will be identification of therapeutic proteolytic agents capable of attacking putative AD pathogens through catalytic degradation of the Ab monomers into non-fibrinogenic and, potentially, anti-amyloid fragments. Our long-term goal is to develop therapeutic strategies, targeting the underlying pathogenic mechanisms in AD.