Pilot Studies of PAX3-FOXO1 Fusions Proteins in Alveolar Rhabdomyosarcoma

项目总结 可用药的人类蛋白质组是巨大的，但只有一小部分是目前FDA批准的靶标 制药公司。这种情况是可以理解的，因为目标疾病相对常见并构成 在人类寿命损失年数、死亡前的生活质量和经济生产力方面的沉重负担。 然而，减轻罕见疾病的负担在伦理和经济上仍有重大需求。为了满足这一点 需要，美国国立卫生研究院发布了RFA-TR-22-030：试点项目，调查与 罕见疾病，我们对此提出了当前的建议。这一试点项目旨在发展生物化学 能够确定PAX3-FOXO1的结构和分子尺度功能的试剂和分析方法 已知的驱动罕见疾病肺泡型横纹肌肉瘤表型的融合蛋白。 在我们对转录因子反式激活结构域的兴趣的推动下，PI的实验室做出了新的贡献 核磁共振波谱技术使转录的高分辨率研究成为可能 因子反式激活结构域及其与下游共调节因子的相互作用，产生了一个独特的机会 为了解决PAX3-FOXO1融合蛋白功能的机制研究尚未得到满足的需求，将使 下游铅化合物筛选和最终药物开发。在这方面，第一个具体目标是 本项目是为了产生重组PAX3-FOXO1融合蛋白的构建并初步完成 融合蛋白DNA结合区和反式激活结构域的结构筛选。跟随 对溶解性和稳定性进行筛选，选定的构建物将通过我们实验室创新的13C进行分析 一种非常适合分析转录因子及其无序的直接检测策略 转录激活域。重要的是，这种融合中包含的FOXO1区域已知受 赖氨酸乙酰化，我们最近开发了生化和光谱技术，使直接 对这些翻译后修饰的研究。第二个具体目标是建立体外功能研究。 研究了PAX3-FOXO1融合蛋白与DNA和蛋白质共调节因子的相互作用。Pax3-FOXO1相互作用 将通过与DNA结合的荧光检测方法和机会筛选靶标进行研究 用于共结晶。由反式激活结构域介导的蛋白质-蛋白质相互作用将使用 沉降速度分析型超速离心法与核磁共振技术的结合 光谱学，以确定在未来的研究中可能被下药的相互作用。这些目标加在一起，将促进 PAX3-FOXO1融合蛋白与罕见疾病表型关系的探讨 泡状横纹肌肉瘤。最终，通过这项初步研究开发的体外系统将被利用 为了确定PAX3-FOXO1融合蛋白如何与合作伙伴相互作用，加深了对分子的理解 为肺泡型横纹肌肉瘤奠定了基础，并为下游高通量文库筛选奠定了基础 能够破坏PAX3-FOXO1反式激活结构域介导的相互作用的先导化合物。

PROJECT SUMMARY The druggable human proteome is vast and yet only a small fraction of it is targeted by current FDA approved pharmaceuticals. This situation is understandable as the diseases targeted are relatively common and pose profound burdens in terms of lost years of human life, quality of life prior to mortality, and economic productivity. Yet there remains a significant ethical and economic need to relieve the burden from rare diseases. To meet this need, the NIH has released RFA-TR-22-030: Pilot Projects Investigating Understudied Proteins Associated with Rare Diseases, to which we respond with the current proposal. This pilot project aims to develop biochemical reagents and assays capable of defining the structure and molecular-scale function of a PAX3-FOXO1 fusion protein that is known to drive the phenotype of the rare disease alveolar rhabdomyosarcoma. Motivated by our interest in transcription factor transactivation domains, the PI's laboratory has contributed novel nuclear magnetic resonance spectroscopy techniques that empower high resolution investigation of transcription factor transactivation domains and their interactions with downstream coregulators, yielding a unique opportunity to address the unmet need for mechanistic studies of PAX3-FOXO1 fusion protein function that will enable downstream lead compound screening and eventual drug development. In this context, the first specific aim of this project is to generation recombinant PAX3-FOXO1 fusion protein constructs and completion of preliminary structure screening for both the fusion protein's DNA binding region and transactivation domain. Following screening for solubility and stability, selected constructs will be analyzed through our laboratory's innovative 13C direct-detect strategy that is uniquely well-suited to analysis of transcription factors and their disordered transactivation domains. Importantly, the region of FOXO1 included in this fusion is known to be regulated by lysine acetylation and we have recently developed biochemical and spectroscopic techniques that enable direct study of these post-translational modifications. The second specific aim is to establish in vitro functional studies of PAX3-FOXO1 fusion protein interactions with both DNA and protein coregulators. PAX3-FOXO1 interactions with DNA will be investigated through fluorescence-detected binding assays and targets of opportunity screened for co-crystallization. Protein-protein interactions mediated by the transactivation domain will be screened using a combination of sedimentation velocity analytical ultracentrifugation and nuclear magnetic resonance spectroscopy to identify interactions that may be druggable in future studies. Together, these aims will facilitate exploration of the relationship between the PAX3-FOXO1 fusion protein and the phenotype of the rare disease alveolar rhabdomyosarcoma. Ultimately, the in vitro system developed through this pilot study will be leveraged to define how the PAX3-FOXO1 fusion protein interacts with partners, advance understanding of the molecular basis for alveolar rhabdomyosarcoma, and set the stage for downstream high-throughput library screening for lead compounds capable of disrupting interactions mediated by the PAX3-FOXO1 transactivation domain.