Chemical probe discovery for PAX3-FOXO1

PAX3-FOXO1 化学探针发现

• 批准号: 10680802
• 负责人: Corinne Mary Linardic
• 金额: $ 19.59万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10680802
• 关键词: Affect; Affinity; Antibodies; Binding; Biological Assay; Cell Line; Cell model; Cells; Cellular Assay; ChIP-seq; Characteristics; Chemicals; Childhood Alveolar Rhabdomyosarcoma; Chimeric Proteins; Chromatin; Chromosomal translocation; Collaborations; Communities; Coupled; DNA; Development; Disease; Engineering; Epitopes; FOXO1A gene; Fusion Oncogene Proteins; Gene Expression; Genetic Transcription; Goals; Label; Laboratories; Length; Ligands; Luciferases; Mass Spectrum Analysis; Oncogenic; PAX3 gene; Patient-Focused Outcomes; Patients; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phenotype; Protac; Proteins; Reporter; Research; Role; Structure; Structure-Activity Relationship; Therapeutic; Toxic effect; Transcript; Validation; analog; biophysical properties; design; drug candidate; experience; genetic approach; improved; mouse model; multidisciplinary; new technology; novel; novel strategies; programs; screening; small molecule; therapeutic target; transcription factor; transcriptome sequencing; tumor; Affect; Affinity; Antibodies; Binding; Biological Assay; Cell Line; Cell model; Cells; Cellular Assay; ChIP-seq; Characteristics; Chemicals; Childhood Alveolar Rhabdomyosarcoma; Chimeric Proteins; Chromatin; Chromosomal translocation; Collaborations; Communities; Coupled; DNA; Development; Disease; Engineering; Epitopes; FOXO1A gene; Fusion Oncogene Proteins; Gene Expression; Genetic Transcription; Goals; Label; Laboratories; Length; Ligands; Luciferases; Mass Spectrum Analysis; Oncogenic; PAX3 gene; Patient-Focused Outcomes; Patients; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phenotype; Protac; Proteins; Reporter; Research; Role; Structure; Structure-Activity Relationship; Therapeutic; Toxic effect; Transcript; Validation; analog; biophysical properties; design; drug candidate; experience; genetic approach; improved; mouse model; multidisciplinary; new technology; novel; novel strategies; programs; screening; small molecule; therapeutic target; transcription factor; transcriptome sequencing; tumor

ABSTRACT – Project 3: Chemical probe discovery for PAX3-FOXO1 The majority of pediatric alveolar rhabdomyosarcoma (ARMS) cases are characterized by a chromosomal translocation encoding the PAX3-FOXO1 fusion oncoprotein, a transcription factor correlated with poor patient outcomes. The overarching goal of this FusOnc2 Center is to advance the therapeutic tractability of the PAX3- FOXO1 fusion protein in ARMS by comprehensively identifying the druggable co-regulators, modulators, and intrinsic activities of PAX3-FOXO1. Transcription factor fusion proteins such as PAX3-FOXO1 have been validated through genetic approaches as key therapeutic targets, since they may represent tumor-specific Achilles’ heels. Unfortunately, to date there are no small molecules that can modulate the function of PAX3- FOXO1. Hence, novel approaches are needed to develop chemical probes or drugs that can target this fusion oncoprotein. Similar to other fusion transcription factors, PAX3-FOXO1 is intrinsically disordered and lacks the traditional small-molecule binding pockets observed in historically tractable targets, complicating structure-driven design or small-molecule screening efforts to identify drug candidates. This Project’s objective is to develop chemical probes for PAX3-FOXO1 using a novel emerging strategy developed within the Koehler Laboratory against other transcription factors and recalcitrant targets. The strategy involves the use of high-throughput and unbiased binding assays involving small-molecule microarrays (SMMs) screened with purified, full-length transcription factor or transcription factor residing in cell lysates. We will exploit this novel technology to develop chemical probes against PAX3-FOXO1 as the first step in drugging this protein through the following four Specific Aims: 1) Execute binding assays for PAX3-FOXO1 using SMMs containing >55,000 small molecules; 2) Perform phenotypic and biophysical characterization of putative PAX3-FOXO1 binders; 3) Synthetically optimize chemical probes and evaluate target engagement in cells; 4) Conduct mechanistic studies of chemical probes in cellular and murine models, in collaboration with the Validation Core. This Project will establish gross structure- activity relationships for 1-3 compounds and use principles of medicinal chemistry to develop new analogs with improved potency in cellular assays and physicochemical characteristics and will synthesize proteolysis targeting chimeras (PROTACs) in an effort to explore targeted degradation of the PAX3-FOXO1 fusion protein. While the aims of this multi-disciplinary proposal are ambitious, the Project will be enabled by the cumulative experience of the team, as well as the extensive capabilities of our FusOnC2 Center and its Projects and Cores. Our approach will lead to high-quality probes of PAX3-FOXO1 function that may clarify the role of the fusion as a direct therapeutic target for ARMS. Through these efforts we also expect to define general strategies for targeting other recalcitrant oncogenic fusion proteins with small molecules.

摘要 - 项目3：PAX3-FOXO的化学探针发现 大多数儿科牙槽横纹肌肉瘤（ARM）病例的特征是染色体 易位编码PAX3-FOXO1融合癌蛋白，转录因子与差的患者相关 结果。该Fusonc2中心的总体目标是提高PAX3-的治疗性障碍。 FOXO1融合蛋白在手臂中通过全面识别可药物的共同调节剂，调节剂和 PAX3-FOXO1的内在活性。转录因子融合蛋白（如Pax3-福克斯1）已经 通过遗传方法作为关键治疗靶标的验证，因为它们可能代表肿瘤特异性 阿喀琉斯高跟鞋。不幸的是，迄今为止，没有小分子可以调节PAX3-的功能 FOXO1。因此，需要新的方法来开发可以瞄准这种融合的化学问题或药物 癌蛋白。与其他融合转录因子类似，Pax3-Foxo1本质上是无序的，缺乏 传统的小分子结合口袋在历史上可牵引的目标中观察到，结构驱动的复杂 设计或小分子筛查工作以识别候选药物。该项目的目标是发展 PAX3-FOXO1的化学问题使用Koehler实验室中开发的新型新兴策略 针对其他转录因子和顽固靶标。该策略涉及使用高通量和 公正的结合测定法涉及纯净的，全长的小分子微阵列（SMM） 驻留在细胞裂解物中的转录因子或转录因子。我们将利用这项新技术来发展 针对Pax3-Foxo1的化学问题是通过以下四个特异性吸毒该蛋白的第一步 目的：1）使用含有> 55,000个小分子的SMM对PAX3-FOXO1进行结合测定； 2）执行 推定的Pax3-FoxO1粘合剂的表型和生物物理表征； 3）合成优化 化学问题并评估细胞中的目标参与； 4）进行化学问题的机械研究 在蜂窝和鼠模型中，与验证核心合作。该项目将建立总体结构 - 1-3种化合物的活动关系，并使用医学化学原理来开发新的类似物 提高了细胞测定和物理特征的效力，并将构成蛋白解靶向靶向 嵌合体（Protac）努力探索PAX3-FOXO1融合蛋白的靶向降解。而 这项多学科提议的目的是雄心勃勃的，该项目将通过累积经验来实现 该团队以及我们的Fusonc2中心及其项目和核心的广泛功能。我们的 方法将导致PAX3-FOXO1功能的高质量问题，该功能可能会阐明融合的作用 直接的武器治疗靶标。通过这些努力，我们还希望定义针对目标的一般策略 其他顽固的致癌融合蛋白，带有小分子。