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

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-FOXO1化学探针的发现 大多数儿童肺泡型横纹肌肉瘤(ARMS)病例的特征是染色体 编码与贫困患者相关的转录因子PAX3-FOXO1融合癌蛋白的易位 结果。这个FusOnc2中心的首要目标是提高PAX3的治疗可操作性- Foxo1融合蛋白在ARM中通过全面识别可药物的辅助调节因子、调节器和 PAX3-FOXO1的内在活性。PAX3-FOXO1等转录因子融合蛋白已被 通过基因方法作为关键治疗靶点进行验证，因为它们可能代表肿瘤特异性 阿喀琉斯的脚跟。不幸的是，到目前为止，还没有小分子可以调节PAX3的功能- 狐狸1号。因此，需要新的方法来开发能够针对这种融合的化学探针或药物 癌蛋白。与其他融合转录因子类似，PAX3-FOXO1在本质上是无序的，缺乏 在历史上容易处理的靶中观察到传统的小分子结合口袋，使结构驱动复杂化 设计或小分子筛选的努力，以确定候选药物。该项目的目标是开发 使用克勒实验室开发的一种新策略对PAX3-FOXO1进行化学探针 对抗其他转录因子和顽固性靶标。该战略涉及使用高吞吐量和 用纯化的全长筛选的小分子微阵列(SMM)的无偏结合分析 转录因子或存在于细胞裂解物中的转录因子。我们将利用这项新技术来开发 针对PAX3-FOXO1的化学探针是通过以下四种特异性药物抑制该蛋白的第一步 目标：1)使用含有55,000个小分子的SMM对PAX3-FOXO1进行结合分析；2)执行 PAX3-FOXO1粘结剂的表型和生物物理特性；3)综合优化 化学探针和评价细胞内靶点的结合；4)化学探针的机理研究 在细胞和小鼠模型中，与验证核心合作。本项目将建立总结构- 1-3类化合物的活性关系及利用药物化学原理开发新的类似物 提高了细胞分析和理化特性的效力，并将合成蛋白水解靶向 嵌合体(PROTAC)，以努力探索PAX3-FOXO1融合蛋白的靶向降解。而当 这一多学科提案的目标是雄心勃勃的，该项目将通过积累的经验来实现 以及我们的FusonC2中心及其项目和核心的广泛能力。我们的 该方法将导致PAX3-FOXO1功能的高质量探针，可能阐明融合作为一种 武器的直接治疗靶点。通过这些努力，我们还期望为目标确定一般战略。 其他顽固性致癌融合蛋白与小分子。