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 化学探针发现 大多数儿科腺泡型横纹肌肉瘤 (ARMS) 病例的特点是染色体异常 编码 PAX3-FOXO1 融合癌蛋白的易位，这是一种与贫困患者相关的转录因子 该 FusOnc2 中心的首要目标是提高 PAX3- 的治疗可处理性。 通过全面鉴定可药物化的协同调节剂、调节剂和 PAX3-FOXO1 等转录因子融合蛋白的内在活性已被证实。 通过遗传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 不幸的是，迄今为止还没有可以调节 PAX3- 功能的小分子。 因此，需要新的方法来开发能够靶向这种融合的化学探针或药物。 与其他融合转录因子类似，PAX3-FOXO1 本质上是无序的并且缺乏 在历史上易于处理的目标中观察到的传统小分子结合口袋，使结构驱动变得复杂 该项目的目标是开发设计或小分子筛选工作来确定候选药物。 使用 Koehler 实验室开发的新型新兴策略开发 PAX3-FOXO1 化学探针 针对其他转录因子和顽固靶标的策略涉及使用高通量和 涉及使用纯化全长筛选的小分子微阵列 (SMM) 的无偏结合测定 我们将利用这项新技术来开发转录因子或存在于细胞裂解物中的转录因子。 针对 PAX3-FOXO1 的化学探针作为通过以下四种特定方式对该蛋白质进行药物治疗的第一步 目标：1) 使用含有 >55,000 个小分子的 SMM 执行 PAX3-FOXO1 结合测定；2) 执行 假定的 PAX3-FOXO1 结合物的表型和生物物理表征；3) 合成优化 化学探针并评估细胞中的靶点参与；4) 进行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，该项目将建立总体结构。 1-3 种化合物的活性关系，并利用药物化学原理开发新的类似物 提高细胞测定和理化特性的效力，并将合成蛋白水解靶向 嵌合体（PROTAC）致力于探索 PAX3-FOXO1 融合蛋白的靶向降解。 这个多学科提案的目标雄心勃勃，该项目将通过积累的经验来实现 团队的实力，以及我们 FusOnC2 中心及其项目和核心的广泛能力。 该方法将产生 PAX3-FOXO1 功能的高质量探针，这可能会阐明融合作为 通过这些努力，我们还期望确定针对 ARMS 的直接治疗靶点。 其他具有小分子的顽固性致癌融合蛋白。