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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.

项目总结融合转录因子(TF)是一类诱人的癌症靶点。当出现在恶性肿瘤中时，这些嵌合蛋白通常是主要的致癌因素，而融合因子的基因敲除通常导致导致细胞死亡或分化。然而，融合因子是一类特别难以靶向的蛋白质。小分子抑制剂。靶向融合因子的首要挑战是显著程度的内在个体融合的无序和缺乏机械性特征，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。支持直接靶向融合因子的策略因此，在不需要对融合因子进行广泛的功能鉴定的情况下实现其功能是非常有价值的。在这项提议中，我将开发可推广的和机械上不偏不倚的方法来发现抑制剂融合因子PAX3-FOXO1采用小分子芯片(SMM)筛选策略。通过培训计划在此详述，我将获得几个化学生物学策略方面的专业知识，这些策略对于实现这个重要的目标。拟议的研究将因特殊的培训环境而得到进一步促进这为我提供了癌症生物学和转铁蛋白抑制剂开发方面的科学带头人的指导。初步数据表明，PAX3-FOXO1是可连接的；一个小的概念验证屏幕产生了两个选择性打击分子，其中之一被证实直接结合PAX3-FOXO1并抑制其在血管内皮细胞中的功能后续化验。在这个初步数据的基础上，我假设了直接靶向PAX3-FOXO1或通过联合分析细胞裂解物和细胞裂解物中PAX3-FOXO1的SMM筛选，可以确定其结合伙伴纯化的蛋白质格式。在目标1中，我将鉴定PAX3-FOXO1的功能活性直接结合物，并鉴定一系列最强大分子的结合部位。其中一个位点的结构特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，这将作为基础用于未来基于结构的优化工作。在目标2中，我将识别与功能结合的活性分子易受攻击的PAX3-FOXO1交互伙伴。使用光亲和标记进行详细的目标识别工作方法将识别分子靶标和铅分子的结合位置，并确认靶标实验将表征目标蛋白在PAX3-FOXO1驱动的转录中的功能。在目标3中，我将通过鉴定高亲和力开发针对嵌合体的PAX3-FOXO1蛋白水解物(PROTAC)降解物 Pax3-FOXO1结合剂并对它们进行有效的筛选和优化策略以确定 PAX3-FOXO1粘合剂、连接剂和E3连接酶粘合剂的组合可导致最有效的降解。这 Pax3-FOXO1 PROTAC，代表着任何融合因子的第一个靶向降解物，将被广泛被定性，并作为化学探头提供给研究界。可概括的和本文概述的无偏见的方法将使我们能够获得关于聚变TF漏洞的新见解同时开发针对这些弱点的化学探测器。