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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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要丰富的血清抗蛋白酶 Alpha-1 抗胰蛋白酶的主要缺陷 E342K 突变体或“Z 变体” (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病和肝病的主要原因。由于 -sheet A 结构域变宽，这种易错组装的变体被认为是高度聚合的它使 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生成肝细胞中积聚导致慢性肝病，并在细胞外积聚导致肺部毒性功能的增强同时伴随着血清 AAT-Z 抗蛋白酶活性的丧失，从而导致中性粒细胞蛋白酶（如弹性蛋白酶）对肺实质的蛋白水解破坏。晶体结构单体 AAT-Z 已经被研究过，之前的突变分析已经证明了其能力该蛋白质表面可及的疏水口袋内的空间填充突变以防止聚合不消除抗蛋白酶活性。因此有人推测小分子可能是发现其作为药理学伴侣，防止 AAT-Z 聚合物形成，同时允许稳定单体的天然抗蛋白酶活性，有助于改善多器官损伤（包括肺病理学）与 AATD 相关。虽然之前已经报道过 AAT-Z 的稳定配体，这些配体普遍无法发挥 AAT-Z 的天然抗蛋白酶活性。在这个培训计划中，我建议使用“全功能化片段”（FFF）子结构以及光交联、亲和力目标 1 中的色谱法和串联质谱法鉴定 AAT-Z 上的小分子位点单体易于被药物样亚结构结合。在目标 2 中，我将开发一种新颖的筛选方法我设想用于识别 AAT-Z 稳定配体或防止 RCL 插入的药理学伴侣同时允许配体结合的 AAT-Z 具有抗蛋白酶活性。在目标 3 中，我将采用基于细胞的表型 Balch 实验室的合作者已经开发并验证了检测方法，可同时评估筛选命中以恢复功能性单体 AAT-Z 分泌效率的能力，同时减少 AATD 培养的肝细胞和肺细胞模型中的细胞内寡聚物。该项目将提供在蛋白质错误折叠生物学领域专家的指导下拥有多学科培训经验，化学蛋白质组学、高通量检测开发以及 AATD 患者治疗。通过在此提出的研究，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，哺乳动物细胞疾病模型的筛选测定优化和应用。这些有价值的以发现为导向的研究能力，以及我之前的临床前培训背景医学和药物化学将有助于实现作为领先医生的高产未来职业生涯一流药物发现这一具有挑战性领域的科学家研究员。