Chemical probe discovery for PAX3-FOXO1

摘要-项目3：PAX 3-FOXO 1的化学探针发现 大多数儿童腺泡状横纹肌肉瘤（ARMS）病例的特征是染色体异常。 编码PAX 3-FOXO 1融合癌蛋白的易位，一种与贫困患者相关的转录因子 结果。FusOnc 2中心的首要目标是提高PAX 3的治疗可处理性， FOXO 1融合蛋白在ARMS中通过全面鉴定可药用的辅助调节剂，调节剂， PAX 3-FOXO 1的内在活性。转录因子融合蛋白如PAX 3-FOXO 1已经被研究。 通过遗传方法验证为关键治疗靶点，因为它们可能代表肿瘤特异性 阿喀琉斯之踵。不幸的是，到目前为止，还没有小分子可以调节PAX 3的功能。 FOXO 1.因此，需要新的方法来开发可以靶向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，PAX 3-FOXO 1本质上是无序的，并且缺乏与转录因子的相互作用。 传统的小分子结合口袋中观察到的历史上听话的目标，复杂的结构驱动 设计或小分子筛选工作，以确定候选药物。该项目的目标是开发 使用Koehler实验室开发的一种新的新兴策略的PAX 3-FOXO 1化学探针 针对其他转录因子和非特异性靶点。该策略涉及使用高通量和 无偏结合试验涉及小分子微阵列（SMM）筛选纯化，全长 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 针对PAX 3-FOXO 1的化学探针作为通过以下四个特异性途径给这种蛋白质下药的第一步， 目的：1）使用含有> 55，000个小分子的SMM执行PAX 3-FOXO 1的结合测定; 2）执行PAX 3-FOXO 1的结合测定。 推定的PAX 3-FOXO 1结合剂的表型和生物物理表征; 3）合成优化 化学探针和评估细胞中的靶接合; 4）进行化学探针的机制研究 在细胞和小鼠模型中，与验证核心合作。本项目将建立总体结构- 1-3种化合物的活性关系，并使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），以努力探索PAX 3-FOXO 1融合蛋白的靶向降解。而 这个多学科的建议目标是雄心勃勃的，该项目将通过积累经验， 以及我们的FusOnC 2中心及其项目和核心的广泛能力。我们 这种方法将导致PAX 3-FOXO 1功能的高质量探针，这可能会阐明融合作为一种免疫调节剂的作用。 ARMS的直接治疗目标。通过这些努力，我们还期望确定确定目标的总体战略 其他与小分子的非致癌性致癌融合蛋白。

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.