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Targeting the Oncogenic Fusion Transcription Factor PAX3-FOXO1 with Small Molecules

用小分子靶向致癌融合转录因子 PAX3-FOXO1

基本信息

批准号：
10372971
负责人：
Matthew James Henley
金额：
$ 1.17万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-01 至 2022-05-06
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10372971
关键词：
Address Affect Affinity Alveolar Rhabdomyosarcoma Binding Binding Sites Biological Biological Assay Biology Biophysics Cancer Biology Cell Death Cell Differentiation process Cells Chemicals Chimeric Proteins Communities Complex Consumption Coupled Data Development Disease Dissection Environment FOXO1A gene Future Genes Genetic Genetic Transcription Goals Individual Joints Knowledge Lead Malignant Childhood Neoplasm Malignant Neoplasms Mentorship Methods Molecular Molecular Target Nature Oncogenic PAX3 gene Photoaffinity Labels Prognosis Protac Proteins Proteome Reporting Research Resistance Series Site Structure Tertiary Protein Structure Testing Therapeutic Time Training Validation base drug discovery experimental study follow-up gene translocation inhibitor insight knock-down novel novel therapeutics screening small molecule small molecule inhibitor targeted treatment therapeutic target transcription factor ubiquitin-protein ligase

项目摘要

PROJECT SUMMARY Fusion transcription factors (TFs) are an enticing class of cancer targets. When present in a malignancy, these chimeric proteins are often the primary oncogenic drivers, and genetic knockdown of the fusion TF often leads to cell death or differentiation. However, fusion TFs are an exceptionally difficult class of proteins to target with small molecule inhibitors. Chief among the challenges of targeting fusion TFs is the significant degree of intrinsic disorder and the lack of mechanistic characterization of individual fusions, limiting the ability to execute structure and/or function-driven approaches to developing inhibitors. Strategies that enable direct targeting of fusion TF function without the need for extensive functional characterization of the fusion TF are therefore highly valuable. In this proposal, I will develop generalizable and mechanistically unbiased approaches to discover inhibitors of the fusion TF PAX3-FOXO1 using small molecule microarray (SMM) screening strategies. Via the training plan detailed herein, I will acquire expertise in several chemical biology strategies that are critical to accomplishing this important objective. The proposed research will be further facilitated by an exceptional training environment that provides me with mentorship from scientific leaders in cancer biology and TF inhibitor development. Preliminary data indicates that PAX3-FOXO1 is ligandable; a small proof-of-concept screen produced two selective hit molecules, one of which was confirmed to directly bind PAX3-FOXO1 and inhibit its function in follow-up assays. Building on this preliminary data, I hypothesize molecules that directly target PAX3-FOXO1 or its binding partners can be identified via joint analysis of SMM screens against PAX3-FOXO1 in cell lysate and purified protein formats. In Aim 1, I will identify functionally active direct binders of PAX3-FOXO1 and identify binding sites for a series of the most potent molecules. Structural characterization of one of these sites and its interactions with binders will provide critical insights about small molecule recognition that will serve as the basis for future structure-based optimization efforts. In Aim 2, I will identify active molecules that bind to functionally vulnerable PAX3-FOXO1 interaction partners. Detailed target identification efforts using photo-affinity labeling approaches will identify the molecular target and binding site of a lead molecule, and target validation experiments will characterize the function of the target protein in PAX3-FOXO1-driven transcription. In Aim 3, I will develop a PAX3-FOXO1 proteolysis targeting chimaera (PROTAC) degrader by identifying high-affinity PAX3-FOXO1 binders and subjecting them to an efficient screening and optimization strategy to identify the combination of PAX3-FOXO1 binder, linker, and E3 ligase binder that leads to the most potent degradation. This PAX3-FOXO1 PROTAC, representing the first targeted degrader of any fusion TF, will be extensively characterized and made available to the research community as a chemical probe. The generalizable and unbiased approaches outlined herein will enable novel insights about fusion TF vulnerabilities to be obtained concurrently with the development of chemical probes that target these weaknesses.

项目摘要融合转录因子（TF）是一类诱人的癌症靶点。当存在于恶性肿瘤中时，这些嵌合蛋白通常是主要的致癌驱动因子，融合TF的基因敲低通常导致细胞死亡或分化。然而，融合TF是一种非常难靶向的蛋白质小分子抑制剂。靶向融合TF的主要挑战是显著程度的内在缺陷。混乱和缺乏个别融合的机械表征，限制了执行结构的能力和/或功能驱动的方法来开发抑制剂。能够直接靶向融合TF的策略因此，不需要融合TF的广泛功能表征的功能是非常有价值的。在这个建议中，我将开发可推广的和机械上无偏见的方法来发现抑制剂，融合TF PAX 3-FOXO 1使用小分子微阵列（SMM）筛选策略。通过培训计划在此详细介绍，我将获得几个化学生物学策略的专业知识，这些策略对实现这一重要目标。一个特殊的培训环境将进一步促进拟议的研究这为我提供了来自癌症生物学和TF抑制剂开发科学领导者的指导。初步数据表明PAX 3-FOXO 1是可配体的;一个小的概念验证筛选产生了两个选择性命中分子，其中之一被证实直接结合PAX 3-FOXO 1，并抑制其功能，后续分析。基于这些初步数据，我假设直接靶向PAX 3-FOXO 1或其结合配偶体可以通过对细胞裂解物中针对PAX 3-FOXO 1的SMM筛选的联合分析来鉴定，纯化的蛋白质形式。在目标1中，我将鉴定PAX 3-FOXO 1的功能活性直接结合剂，并鉴定一系列最有效分子的结合位点。这些位点之一的结构表征及其与粘合剂的相互作用将提供关于小分子识别的关键见解，未来的结构优化工作。在目标2中，我将识别与功能性结合的活性分子易受攻击的PAX 3-FOXO 1相互作用伙伴。使用光亲和标记的详细目标识别工作这些方法将确定先导分子的分子靶点和结合位点，实验将表征靶蛋白在PAX 3-FOXO 1驱动的转录中的功能。在目标3中，我将开发PAX 3-FOXO 1蛋白水解靶向嵌合体（PROTAC）降解剂， PAX 3-FOXO 1结合剂，并使它们经受有效的筛选和优化策略以鉴定PAX 3-FOXO 1结合剂。 PAX 3-F0 X 01结合剂、接头和E3连接酶结合剂的组合，其导致最有效的降解。这 PAX 3-FOXO 1 PROTAC代表了任何融合TF的第一个靶向降解剂，将被广泛应用于表征并作为化学探针提供给研究界。可推广的和本文概述的无偏方法将使人们能够获得有关融合TF脆弱性的新见解同时开发针对这些弱点的化学探针。