Allosteric Pharmacologic Chaperones for alpha-1 Antitrypsin Mutants

项目摘要/摘要 富含血清抗蛋白酶α-1抗胰蛋白酶的E342K突变体或“Z变异体”的主要缺陷 (AAT)是与Alpha-1抗胰蛋白酶相关的绝大多数发病率和死亡率的原因 缺乏症(AATD)是影响全球数百万患者的遗传性肺和肝脏疾病的主要原因。 由于扩大了-Sheet A结构域，这种易错失的变体已知是高度聚合的 这使AAT-Z单体易于形成细胞毒性环片低聚物。这些有毒的低聚物 在产生肝细胞时积聚，导致慢性肝病，并在细胞外积聚，导致 这两种中毒功能的获得都伴随着血清AAT-Z抗蛋白酶活性的丧失，从而导致 中性粒细胞类弹性蛋白酶对肺实质的蛋白水解性破坏。晶体结构的研究 已对单体AAT-Z进行了研究，先前的突变分析表明， 在该蛋白质表面可接近的疏水口袋内填充空间突变以防止聚合 而不会丧失抗蛋白酶活性。因此，人们假设小分子可能是 发现了作为药理伴侣的化合物，在允许的同时防止AAT-Z聚合物的形成 稳定单体的天然抗蛋白酶活性，用于改善多器官损伤(包括 肺病理)与AATD相关。虽然先前已经报道了AAT-Z的稳定配体， 这些配体普遍不允许AAT-Z具有天然的抗蛋白酶活性。在这份培训计划中，我 建议使用完全功能化的片段(FFF)亚结构以及光交联、亲和力 AIM 1的层析和串联质谱学鉴定AAT-Z上的小分子中心 可通过类药物亚结构结合的单体。在目标2中，我将开发一种新的筛选分析方法 我构思了用于识别AAT-Z稳定配体或阻止RCL插入的药理伴侣 同时允许配体结合的AAT-Z的抗蛋白酶活性。在目标3中，我将采用基于细胞的表型 已经由我们在Balch实验室的合作者开发和验证的测试，以同时评估 筛选HITS以恢复功能性单体AAT-Z分泌效率的能力，同时降低 培养的AATD肝细胞和肺细胞模型中的细胞内寡聚体。这个项目将负担得起 在蛋白质错误折叠生物学领域专家的指导下的多学科培训经验， 化学蛋白质组学，高通量分析的发展，以及AATD患者的治疗。通过 在这里提出的研究，我将在最先进的蛋白质组分析方法方面发展强大的专业知识， 疾病哺乳动物细胞模型的筛选、检测、优化及应用。这些贵重物品 以发现为导向的研究能力，加上我之前在临床前的培训背景 医学和药物化学将有助于实现作为一名领先医生的高生产率的未来职业生涯- 在具有挑战性的一流药物发现领域的科学家研究员。

Project Summary/Abstract The major deficiency E342K mutant or “Z-variant” of the abundant serum antiprotease Alpha-1 antitrypsin (AAT) is responsible for the vast majority of morbidity and mortality associated with Alpha-1 antitrypsin deficiency (AATD), a leading cause of hereditary lung and liver disease affecting millions of patients globally. This missasembly-prone variant is known to be highly polymerogenic, owing to a widened -sheet A domain which predisposes the AAT-Z monomer to form cytotoxic loop-sheet oligomers. These toxic oligomers accumulate in producing hepatocytes leading to chronic liver disease, and build up extracellularly leading to both gain-of-toxic function in the lung with a concomitant loss of serum AAT-Z antiprotease activity which leads to proteolytic destruction of lung parenchyma by neutrophil proteases like elastase. Crystal structures of monomeric AAT-Z have been studied and previous mutational analyses have demonstrated the capacity for space-filling mutations within surface-accessible hydrophobic pockets on this protein to prevent polymerization without abrogating antiprotease activity. It has thus been hypothesized that small molecules could be discovered which act as pharmacological chaperones, preventing AAT-Z polymer formation while permitting native antiprotease activity of the stabilized monomer, serving to ameliorate the multiorgan injury (including lung pathology) associated with AATD. While stabilizing ligands for AAT-Z have previously been reported, these ligands universally fail to permit the native antiprotease activity of AAT-Z. In this training proposal, I propose to use Fully-Functionalized Fragment” (FFF) substructures along with photo-crosslinking, affinity chromatography and tandem mass-spectrometry in Aim 1 to identify the small molecule sites on the AAT-Z monomer accessible to binding by drug-like substructures. In Aim 2 I will develop a novel screening assay that I conceived of for identifying AAT-Z stabilizing ligands or pharmacologic chaperones that prevent RCL insertion while permitting antiprotease activity of ligand-bound AAT-Z. In Aim 3 I will employ a cell-based phenotypic assay already developed and validated by our collaborators in the Balch Lab to simultaneously evaluate the capacity for screening hits to restore functional monomeric AAT-Z secretion efficiency, while reducing intracellular oligomers in cultured hepatocyte and pulmonary cell models of AATD. This project will afford a multidisciplinary training experience with guidance from experts in the fields of protein misfolding biology, chemical proteomics, high-throughput assay development, as well as AATD patient treatment. Through the research proposed herein, I will develop a robust expertise in state-of-the-art methods for proteomic analysis, screening assay optimization, and applications of mammalian cell models of disease. These valuable discovery-oriented research competencies, together with my previous training background in pre-clinical medicine and medicinal chemistry will serve to enable a highly productive future career as a leading physician- scientist researcher in the challenging field of first-in-class drug discovery.