Allosteric Pharmacologic Chaperones for alpha-1 Antitrypsin Mutants

α-1 抗胰蛋白酶突变体的变构药理学伴侣

• 批准号: 10633070
• 负责人: Adrian M. Guerrero
• 金额: $ 3.47万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10633070
• 关键词: Address; Adult; Affect; Affinity; Affinity Chromatography; Alkynes; Binding; Binding Sites; Biological Assay; Biology; Cell model; Cells; Chemicals; Chemistry; Chronic; Chronic Obstructive Pulmonary Disease; Clinical; Clinical Pathology; Competence; Complex; Crystallography; Disease model; Elastases; Enzyme-Linked Immunosorbent Assay; Exhibits; Future; Hepatocyte; Homeostasis; Hydrophobicity; Impairment; In Situ; In Vitro; Inherited; Institution; Laboratories; Libraries; Ligand Binding; Ligands; Literature; Liver; Liver Failure; Liver diseases; Lung; Lung diseases; Mammalian Cell; Maps; Mass Spectrum Analysis; Measures; Medicine; Methods; Modeling; Molecular Chaperones; Morbidity - disease rate; Mutation; Mutation Analysis; Organ; Pathology; Patients; Peptide Hydrolases; Peptides; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phenotype; Photoaffinity Labels; Physicians; Polymers; Productivity; Protease Inhibitor; Proteins; Proteome; Proteomics; Proxy; Pulmonary Pathology; Reaction; Reporting; Research; Research Personnel; Role; Scientist; Serum; Site; Structure; Structure of parenchyma of lung; Surface; System; Therapeutic; Training; Trypsin; Variant; alpha 1-Antitrypsin; alpha 1-Antitrypsin Deficiency; assay development; career; cellular pathology; chemoproteomics; chronic liver disease; cytotoxic; cytotoxicity; drug discovery; early onset; experience; extracellular; functional restoration; high throughput screening; in silico; in vivo; innovation; loss of function; miniaturize; monomer; mortality; multidisciplinary; multiorgan injury; mutant; neutrophil; novel; peptidomimetics; pharmacologic; plasma protein Z; polymerization; pre-clinical; preservation; prevent; protein misfolding; screening; small molecule; tandem mass spectrometry; trafficking

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.

项目总结/摘要 丰富的血清抗蛋白酶α-1抗胰蛋白酶的主要缺陷E342 K突变体或“Z-变体” (AAT)是与α-1抗胰蛋白酶相关的绝大多数发病率和死亡率的原因 AATD是遗传性肺病和肝病的主要原因，影响全球数百万患者。 这种容易错配的变体已知是高度聚合的，这是由于加宽的折叠A结构域 其使AAT-Z单体易于形成细胞毒性环片寡聚体。这些有毒的低聚物 在产生肝细胞中积累，导致慢性肝病，并在细胞外积累， 肺中毒性功能的获得和伴随的血清AAT-Z抗蛋白酶活性的丧失， 涉及中性粒细胞蛋白酶如弹性蛋白酶对肺实质的蛋白水解破坏。晶体结构 已经研究了单体AAT-Z，并且先前的突变分析已经证明了 这种蛋白质表面可接近的疏水口袋内的空间填充突变，以防止聚合 而不消除抗蛋白酶活性。因此，有人假设小分子可以是 发现其作为药理学伴侣，阻止AAT-Z聚合物形成，同时允许 稳定单体的天然抗蛋白酶活性，用于改善多器官损伤（包括 肺病理学）与AATD相关。虽然先前已经报道了用于AAT-Z的稳定配体， 这些配体普遍不能使AAT-Z具有天然的抗蛋白酶活性。在这个培训计划中，我 建议使用“全功能化片段”（FFF）子结构，沿着光交联、亲和 AAT-Z上的小分子位点， 单体可通过药物样亚结构结合。在目标2中，我将开发一种新的筛选测定法， 我设想了一种用于鉴定AAT-Z稳定配体或防止RCL插入的药理学伴侣的方法， 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 我们在Balch实验室的合作者已经开发和验证了该检测方法， 筛选命中以恢复功能性单体AAT-Z分泌效率的能力，同时降低 AATD的培养肝细胞和肺细胞模型中的细胞内寡聚体。该项目将提供一个 在蛋白质错误折叠生物学领域专家的指导下， 化学蛋白质组学、高通量检测开发以及AATD患者治疗。通过 在这里提出的研究，我将发展一个强大的专业知识，在国家的最先进的方法，蛋白质组学分析， 筛选测定优化和疾病的哺乳动物细胞模型的应用。这些宝贵 以发现为导向的研究能力，加上我以前在临床前的培训背景， 医学和药物化学将有助于使一个高生产力的未来职业生涯作为一个领先的医生- 科学家研究员在具有挑战性的领域的一流的药物发现。