Probing the Proteinopathy Component of Light Chain Amyloidosis Pharmacologically

项目摘要 免疫球蛋白轻链淀粉样变性(AL)是一种由轻链引起的退行性疾病 (LC)错误折叠和聚集。全长(FL)LC由克隆扩增的浆细胞分泌 AL的人口。因此，AL患者同时患有浆细胞癌和LC错误折叠，并 聚集相关蛋白病，似乎损害器官功能，导致进行性 器官退化。目前，AL是通过改变用途的多发性骨髓瘤药物来治疗的，这种药物可以杀死克隆血浆 分泌FL LCS的细胞。需要机械上不同的治疗方法，特别是对患者来说。 心脏受累，不能耐受目前可用的化疗方案。在这份提案中， 我们寻求生产最终能够在FL LC动力稳定器上进行临床试验的候选药物 靶向AL的蛋白病变成分。在特定目标1中，我们假设LC动力学稳定剂 在我们的Hit-to-Lead药物化学努力中合成可以分解为四个子结构- “锚子结构”、“芳香族核心”、“连接体模块”和“末端芳香族组分”。这 假设是基于迄今已解决的11(FL LC)2·动力学稳定剂晶体结构和结构-- 过去18年合成300多个FL-LC_2动力学稳定剂的活性关系数据 月份。我们将继续使用我们在开发药物时学到的基于结构的设计原则 除了计算工具，特别是Schrödinger LiveDesign软件之外，taamiidis还可以取代 代谢不稳定和潜在毒性的香豆素芳香族核心和二乙基苯胺锚定亚结构 在我们的LC动力学稳定剂中，引入了减少白蛋白结合的官能团。LiveDesign软件 利用预测的LogP值和停靠分数的加权组合来准确预测白蛋白 有约束力的。在特定的目标2中，我们将建立FL LC_2等离子体结合选择性分析来验证我们的 减少白蛋白结合的计算努力。首先，我们将添加荧光标记的FL淀粉样变性 LCS混合健康供体血浆，以及蛋白酶K和候选动力学稳定剂，并如下 随着时间的推移，蛋白水解酶K内切作用与FL LC2构象漂移有关。如果 动力学稳定剂候选者选择性地将导致淀粉样变的FL LC2结合在所有其他血浆蛋白上， 包括白蛋白、FL、LC2、蛋白水解酶K在内的内切蛋白都会被阻止--这一检测方法在很大程度上得到了发展。 下一步，将建立一种亚基交换试验来定量测定药物FL LC2在体内的动力学稳定性。 人体血浆。该检测还可以定量动力学稳定剂与白蛋白的结合。一种荧光标记的 Cys214Ser FL LC2变异体促进亚基交换实验，提供动力学稳定剂的Kd 与添加到健康血浆中的FL LC2变体的结合，以及用于结合到 血浆中的内源性人白蛋白。这些Kd将被用来产生结合选择性比率，即 动力学稳定剂结合到白蛋白/KD的动力学稳定剂结合到FL LC2，我们将最大化这一点。

Project Summary Immunoglobulin light chain amyloidosis (AL) is a degenerative disease that putatively arises from light chain (LC) misfolding and aggregation. Full-length (FL) LCs are secreted from a clonally expanded plasma cell population in AL. Thus, AL patients suffer from both a plasma cell cancer and a LC misfolding and aggregation-associated proteinopathy that appears to compromise organ function, leading to progressive organ deterioration. Currently, AL is treated by repurposed multiple myeloma drugs that kill the clonal plasma cells secreting FL LCs. Mechanistically distinct therapeutic approaches are needed, particularly for patients with cardiac involvement who cannot tolerate the currently available chemotherapy regimens. In this proposal, we seek to produce lead candidates that ultimately would enable a clinical trial on a FL LC kinetic stabilizer targeting the proteinopathy component of AL. In Specific Aim 1, we hypothesize that the LC kinetic stabilizers to be synthesized in our hit-to-lead medicinal chemistry efforts can be dissected into four substructures—the “anchor substructure”, the “aromatic core”, the “linker module” and the “terminal aromatic component”. This hypothesis is based on 11 (FL LC)2•kinetic stabilizer crystal structures solved to date as well as the structure- activity relationship data resulting from the synthesis of over 300 FL LC2 kinetic stabilizers during the past 18 months. We will continue to use structure-based design principles that we learned developing the drug tafamidis, in addition to computational tools, especially the Schrödinger LiveDesign software, to replace the metabolically unstable and potentially toxic coumarin aromatic core and the diethyl aniline anchor substructure in our LC kinetic stabilizers, while introducing functionality to reduce albumin binding. The LiveDesign software utilizes a weighted combination of the predicted LogP values and docking scores to accurately predict albumin binding. In Specific Aim 2, we will develop FL LC2 plasma binding selectivity assays to validate our computational efforts to diminish albumin binding. First, we will add fluorescently labeled FL amyloidogenic LCs to pooled healthy donor plasma, along with proteinase K and a candidate kinetic stabilizer, and follow proteinase K endoproteolysis linked to FL LC2 conformational excursions chromatographically over time. If the kinetic stabilizer candidate selectively binds the amyloidogenic FL LC2 over all the other plasma proteins, including albumin, FL LC2 proteinase K endoproteolysis will be prevented–this assay is largely developed. Next, a subunit exchange assay will be developed to quantify pharmacologic FL LC2 kinetic stabilization in human plasma. This assay also quantifies kinetic stabilizer binding to albumin. A fluorescently labeled Cys214Ser FL LC2 variant facilitates subunit exchange experiments that afford the KD of kinetic stabilizer binding to the FL LC2 variants added to healthy plasma, as well as the kinetic stabilizer KD for binding to endogenous human albumin in plasma. These KDs will be used to generate binding selectivity ratios, i.e., KD of kinetic stabilizer binding to albumin / KD of kinetic stabilizer binding to the FL LC2, which we will maximize.