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（TFS）是一种诱人的癌症靶标。当出现恶性肿瘤时嵌合蛋白通常是主要的致癌驱动因素，融合TF的遗传敲低通常会导致到细胞死亡或分化。但是，Fusion TF是一类极难的蛋白质类别小分子抑制剂。靶向融合TF的挑战中的主要是固有的重要程度混乱和缺乏单个融合的机械表征，限制了执行结构的能力和/或功能驱动的抑制剂方法。能够直接定位Fusion TF的策略因此，无需进行融合TF的广泛功能表征的功能非常有价值。在此提案中，我将开发可概括且机械上无偏的方法来发现的抑制剂使用小分子微阵列（SMM）筛选策略的Fusion TF PAX3-FOXO1。通过培训计划本文详细说明，我将获得几种化学生物学策略的专业知识，这些策略对于完成至关重要这个重要的目标。拟议的研究将通过出色的培训环境进一步促进这为我提供了癌症生物学和TF抑制剂开发方面的科学领导者的指导。初步数据表明PAX3-FOXO1是可韧的。一个小的概念验证屏幕产生了两个选择性命中分子，其中一种已被确认直接结合pax3-foxo1并抑制其在后续测定。在此初步数据的基础上，我假设直接针对Pax3-Foxo1或可以通过对细胞裂解物中PAX3-FoxO1的SMM筛选的联合分析来确定其结合伴侣纯化的蛋白质格式。在AIM 1中，我将识别Pax3-Foxo1的功能活性直接粘合剂并识别一系列最有效分子的结合位点。这些站点之一及其的结构表征与粘合剂的相互作用将提供有关小分子识别的关键见解，该识别将作为基础用于将来基于结构的优化工作。在AIM 2中，我将确定与功能结合的活性分子脆弱的PAX3-FOXO1相互作用伙伴。使用照片亲和力标签的详细目标识别工作方法将识别铅分子的分子靶标和结合位点，并靶向验证实验将表征靶蛋白在PAX3-FoxO1驱动的转录中的功能。在AIM 3中，我通过识别高亲和力，将开发靶向嵌合体（Protac）降解器的PAX3-福克斯1蛋白水解。 PAX3-FOXO1粘合剂，并对它们进行有效的筛选和优化策略，以识别 PAX3-FOXO1粘合剂，接头和E3连接酶粘合剂的组合，导致最有效的降解。这 PAX3-FOXO1 PROTAC代表任何融合TF的第一个靶向降解器，将是广泛的将研究社区作为化学探测器进行了特征并提供。可推广和本文概述的公正方法将使有关融合tf漏洞的新见解同时与针对这些弱点的化学探针的发展。