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：
10390150
负责人：
Adrian M. Guerrero
金额：
$ 3.38万
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/10390150
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 Medicine Clinical Pathology Competence Complex Crystallization Disease model Elastases Enzyme-Linked Immunosorbent Assay Exhibits Future Hepatocyte Homeostasis Hydrophobicity Impairment In Situ In Vitro Inherited Laboratories Libraries Ligand Binding Ligands Literature Liver Liver Failure Liver diseases Lung Lung diseases Mammalian Cell Maps Mass Spectrum Analysis Measures Methods Modeling Molecular Chaperones Morbidity - disease rate Mutation Mutation Analysis Pathology Patients Peptide Hydrolases Peptides Pharmaceutical Chemistry Pharmaceutical Preparations Pharmacology Phenotype Photoaffinity Labels Physicians Polymers Protease Inhibitor Proteins Proteome Proteomics Proxy Pulmonary Pathology Reporting Research Research Personnel Role Scientist Serum Site Structure Structure of parenchyma of lung Surface System Therapeutic Training Variant alpha 1-Antitrypsin alpha 1-Antitrypsin Deficiency assay development base career cellular pathology chronic liver disease crosslink cytotoxic cytotoxicity drug discovery early onset experience extracellular high throughput screening in silico in vivo innovation loss of function miniaturize monomer mortality multidisciplinary multiorgan injury mutant neutrophil novel peptidomimetics 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.

项目摘要/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变化” （AAT）负责与α-1抗胰蛋白酶相关的绝大多数发病率和死亡率缺乏症（AATD），这是遗传性肺和肝病的主要原因，影响了全球数百万患者。众所周知，这种损坏的容易发生变体具有高度聚合性的作用，这是由于 这使AAT-Z单体易于形成细胞毒性环形寡聚物。这些有毒的低聚物积累产生肝细胞，导致慢性肝病，并在细胞外积累两者都在肺中获得毒性功能，并伴随着血清AAT-Z抗蛋白酶活性的丧失通过弹性蛋白酶等嗜中性粒细胞蛋白酶对肺实质的蛋白水解破坏。晶体结构单体AAT-Z已经研究了，以前的突变分析证明了该蛋白质上可透露疏水口袋内的空间填充突变，以防止聚合不清除抗蛋白酶活性。因此，已经假设小分子可能是发现哪种充当药物伴侣，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，可改善多机器人损伤（包括与AATD相关的肺病理学）。虽然以前已经报道了AAT-Z的稳定配体，但这些配体普遍无法允许AAT-Z的天然抗蛋白酶活性。在这个培训建议中，我使用彻底官能化片段的提案”（FFF）子结构以及照片连接，亲和力 AIM 1中的色谱和串联质谱法，以识别AAT-Z上的小分子位点单体可以通过类似药物的子结构结合。在AIM 2中，我将开发一种新颖的筛选测定法我想识别AAT-Z稳定配体或药物伴侣，以防止RCL插入同时允许结合配体AAT-Z的抗蛋白酶活性。在AIM 3中，我将采用基于细胞的表型我们的合作者在Balch Lab中已经开发和验证了测定，以简单地评估筛选命中的能力恢复功能性单体AAT-Z分泌效率，同时降低 AATD培养的肝细胞和肺细胞模型中的细胞内寡聚物。这个项目将负担得起多学科的培训经验在蛋白质错误折叠生物学领域的专家指导下，化学蛋白质组学，高通量测定开发以及AATD患者治疗。通过本文提出的研究，我将在蛋白质组学分析的最先进方法中开发强大的专业知识，筛选测定优化以及哺乳动物细胞模型的疾病。这些有价值以发现为导向的研究能力，以及我以前的临床前培训背景医学和医学化学将使作为领先的医师的未来生产力高，一流药物发现挑战领域的科学家研究员。