Potent and specific small molecule stabilizers of amyloidogenic antibody light chains

轻链淀粉样变性(AL)是一种全身性退行性疾病，其原因是细胞的错误折叠和聚集 游离抗体轻链(LC)蛋白，从单克隆性浆细胞扩增中分泌。这 这一过程会导致包括淀粉样纤维在内的LC聚集物的积聚，最终导致器官衰竭。 现有的AL治疗方法主要集中在使用细胞毒化疗来消除浆细胞的扩张。 然而，许多患者，特别是那些心脏受累的患者，无法耐受这些治疗方案。 尽管AL治疗取得了进步，但美国每年都有1000名患者在确诊后一年内死亡，而且 目前有12000名美国人患有这种疾病。因此，AL的新疗法是一个紧迫的未得到满足的问题 医疗需要。易于聚集的LC具有较低的运动稳定性，即从 天然折叠，无毒的LC形成聚集能力的构象。稳定分子的小分子 天然状态的易于聚集的蛋白质已显示出临床疗效，例如，动力稳定剂药物 以转甲状腺素蛋白为靶点的他法米司是治疗转甲状腺素淀粉样变性的一线药物。我 设想一种类似的LCS运动稳定剂也会有同样的效果。这一治疗策略 应该耐受性良好，是现有AL疗法的补充。使用高通量筛选，我们 确定了动力学稳定LCS的小分子，减缓了构象激活的异常蛋白分解 短途旅行。这些小分子可能会稳定大多数LC序列，因为我们发现的结合位点是 在AL LC序列中高度保守-至关重要，因为每个AL患者都有唯一的LC序列。然而， 屏幕上的点击显示出较低的M解离常数，这对候选药物来说是不够的。我 假设可以产生高效力(低纳米分子KD)和选择性的小分子来 动态稳定易于聚集的LC。为了验证这一假设，我将使用命中到领先的药物 化学、X射线结晶学和计算机辅助结构设计，以提高效力和 LC动力学稳定剂的选择性。我将使用一种筛选热门药物7-二乙氨基-4-甲基香豆素(1)作为 用于优化的模板。在具体目标1中，我将通过添加一个子结构来提高此命中的效力 这延伸到一个由LC·1络合物的共晶结构揭示的空口袋里。具体而言 目标2，我将使用“脚手架跳跃”的方法来提高效力和消除新陈代谢的负担 香豆素核心和锚定子结构。在具体目标3中，我将评估动力学的效力和选择性 来自AIMS 1和2的稳定剂使用荧光标记的LC蛋白酶敏感性分析 人类血浆，使用序列多样化的与疾病相关的LC。为了在AIMS 1和AIMS 2中进一步优化，我 将使用X射线晶体学来获得动力学稳定剂·LC络合物的结构洞察力，并使用现代 促进基于结构的设计的计算方法。我的提议的主要结果将是 确定其他人将继续进行Lead优化的Lead LC动力学稳定剂。

Light chain amyloidosis (AL) is a systemic degenerative disease caused by the misfolding and aggregation of free antibody light chain (LC) proteins that are secreted from a monoclonal plasma cell expansion. This process results in buildup of LC aggregates, including amyloid fibrils, ultimately leading to organ failure. Existing treatments for AL focus on eradicating the plasma cell expansion using cytotoxic chemotherapy. However, many patients, especially those with cardiac involvement, cannot tolerate these treatment regimens. Despite advances in AL treatment, each year, a thousand patients in the US die within a year of diagnosis, and 12,000 Americans currently live with the disease. Therefore, new treatments for AL represent a pressing unmet medical need. Aggregation-prone LCs have low kinetic stability, i.e. high rates of transient unfolding from the natively folded, nontoxic LC into aggregation-competent conformations. Small molecules which stabilize the native state of aggregation-prone proteins have shown clinical efficacy, e.g., the kinetic stabilizer drug tafamidis that targets the protein transthyretin is a frontline treatment for the transthyretin amyloidoses. I envision that an analogous kinetic stabilizer for LCs would be similarly efficacious. This treatment strategy should be well-tolerated and is complementary to existing AL therapies. Using high-throughput screening, we identified small molecules that kinetically stabilize LCs, slowing aberrant proteolysis enabled by conformational excursions. These small molecules will likely stabilize most LC sequences, as the binding site we discovered is highly conserved in AL LC sequences–critical because each AL patient has a unique LC sequence. However, the hits from the screen exhibit low M dissociation constants, which is insufficient for drug candidates. I hypothesize that highly potent (low nanomolar KD’s) and selective small molecules can be generated to kinetically stabilize aggregation-prone LCs. To test this hypothesis, I will employ hit-to-lead medicinal chemistry, X-ray crystallography, and computer-aided structure-based design to improve the potency and selectivity of the LC kinetic stabilizers. I will use one screening hit, 7-diethylamino-4-methylcoumarin (1), as a template for optimization. In Specific Aim 1, I will improve the potency of this hit by appending a substructure that extends into an unoccupied pocket revealed by the co-crystal structure of the LC•1 complex. In Specific Aim 2, I will use a “scaffold hopping” approach to improve potency and remove the metabolic liabilities of the coumarin core and anchor substructure. In Specific Aim 3, I will assess the potency and selectivity of kinetic stabilizers coming from Aims 1 & 2 using a fluorescently-labeled LC protease sensitivity assay conducted in human plasma, using sequence diverse disease-associated LCs. To inform further optimization in Aims 1 & 2, I will use X-ray crystallography to obtain structural insight into kinetic stabilizer•LC complexes and use modern computational approaches to facilitate structure-based design. The primary outcome of my proposal will be the identification of lead LC kinetic stabilizers that will be carried forward to lead optimization by others.