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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.

项目概要融合转录因子（TF）是一类诱人的癌症靶标。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素，融合 TF 的基因敲除通常会导致导致细胞死亡或分化。然而，融合转录因子是一类异常难以靶向的蛋白质小分子抑制剂。以融合转录因子为目标的主要挑战是内在的显着程度无序和缺乏个体融合的机械特征，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。能够直接靶向融合 TF 的策略因此，无需对融合 TF 进行广泛的功能表征即可获得功能，因此非常有价值。在本提案中，我将开发可推广且机械上无偏见的方法来发现抑制剂使用小分子微阵列 (SMM) 筛选策略进行融合 TF PAX3-FOXO1。通过培训计划在此详细介绍，我将获得对实现目标至关重要的几种化学生物学策略的专业知识这个重要目标。卓越的培训环境将进一步促进拟议的研究这为我提供了癌症生物学和 TF 抑制剂开发领域科学领导者的指导。初步数据表明PAX3-FOXO1是可配体的；一个小的概念验证屏幕产生了两个选择性命中分子，其中一种被证实直接结合 PAX3-FOXO1 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 PAX3-FOXO1 或通过对细胞裂解液中针对 PAX3-FOXO1 的 SMM 筛选进行联合分析，可以识别其结合伙伴纯化的蛋白质形式。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有效的分子的结合位点。这些地点之一的结构特征及其与结合剂的相互作用将提供关于小分子识别的重要见解，这将作为基础为未来基于结构的优化工作。在目标 2 中，我将识别功能性结合的活性分子易受攻击的 PAX3-FOXO1 相互作用伙伴。使用照片亲和标记进行详细的目标识别工作方法将确定分子靶标和先导分子的结合位点，以及靶标验证实验将表征靶蛋白在 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来开发 PAX3-FOXO1 蛋白水解靶向嵌合体 (PROTAC) 降解剂 PAX3-FOXO1 结合物并对它们进行有效的筛选和优化策略以识别 PAX3-FOXO1 结合物、接头和 E3 连接酶结合物的组合，可产生最有效的降解。这 PAX3-FOXO1 PROTAC 代表任何融合 TF 的第一个靶向降解剂，将被广泛应用表征并作为化学探针提供给研究界。可概括的和本文概述的公正方法将能够获得有关融合 TF 漏洞的新颖见解同时开发针对这些弱点的化学探针。