Pilot Studies of PAX3-FOXO1 Fusions Proteins in Alveolar Rhabdomyosarcoma

PAX3-FOXO1 融合蛋白在肺泡横纹肌肉瘤中的初步研究

• 批准号: 10726763
• 负责人: Scott A Showalter
• 金额: $ 15.61万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-15 至 2024-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10726763
• 关键词: Abbreviations; Acetylation; Address; Affect; Affinity; Alveolar Rhabdomyosarcoma; Binding; Binding Sites; Biochemical; Biological Assay; Biology; C-terminal; Cell model; Chemicals; Chimeric Proteins; Chromosomal translocation; Classification; Clinical; Complex; Crystallization; DNA; DNA Binding; DNA-Binding Proteins; Development; Disease; EP300 gene; Economics; Embryo; Enhancers; Ethics; Etiology; FDA approved; FOXO1A gene; Fluorescence; Foundations; Future; Generations; Genes; Genetic Markers; Genetic Transcription; Grant; Human; In Vitro; Individual; Investigation; Knowledge; Laboratories; Lead; Length; Libraries; Life; Lysine; Malignant Childhood Neoplasm; Mediating; Molecular; Myoblasts; NMR Spectroscopy; Outcome; PAX3 gene; Patients; Pharmaceutical Preparations; Pharmacologic Substance; Phenotype; Pilot Projects; Positioning Attribute; Post-Translational Protein Processing; Productivity; Prognosis; Proteins; Proteome; Protocols documentation; Public Health; Quality of life; Rare Diseases; Reagent; Recombinants; Research; Resolution; Rhabdomyosarcoma; Skeletal Muscle; Soft tissue sarcoma; Solubility; Source; Structure; System; Techniques; Testing; Therapeutic; Transactivation; Transcriptional Regulation; Translating; United States; United States National Institutes of Health; analytical ultracentrifugation; biochemical tools; biophysical analysis; chemotherapeutic agent; design; drug development; economic need; empowerment; homeodomain; innovation; insight; interest; molecular scale; mortality; novel; novel therapeutics; patient prognosis; polypeptide; pre-clinical; programs; promoter; protein function; protein protein interaction; recruit; screening; sedimentation velocity; small molecule; structural biology; t(2; 13)(q35; q14); transcription factor; translational potential; treatment arm; tumor

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.

项目摘要 可药用的人类蛋白质组是巨大的，但只有一小部分是目前FDA批准的目标 大药厂这种情况是可以理解的，因为所针对的疾病比较常见， 在人的寿命损失、死亡前的生活质量和经济生产力方面，这是一个沉重的负担。 然而，在伦理和经济上仍然需要减轻罕见疾病的负担。满足这一 需要，NIH已经发布了RFA-TR-22-030：研究与癌症相关的未充分研究的蛋白质的试点项目。 罕见疾病，我们以目前的建议作出回应。该试验项目旨在开发生物化学 能够确定PAX 3-FOXO 1的结构和分子级功能的试剂和测定 融合蛋白，已知其驱动罕见疾病腺泡状横纹肌肉瘤的表型。 出于我们对转录因子反式激活结构域的兴趣，PI实验室提供了新的 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辅助调节因子的相互作用， 为了解决PAX 3-FOXO 1融合蛋白功能的机制研究的未满足的需求， 下游先导化合物筛选和最终药物开发。在这方面，第一个具体目标是 本项目旨在构建重组PAX 3-FOXO 1融合蛋白，并完成初步的 对融合蛋白的DNA结合区和反式激活结构域进行结构筛选。以下 筛选的溶解性和稳定性，选定的结构将通过我们实验室的创新13 C 直接检测策略，其独特地非常适合于分析转录因子及其无序结构， 反式激活结构域重要的是，包括在这种融合中的FOXO 1区域已知是由 赖氨酸乙酰化，我们最近开发了生物化学和光谱技术， 研究这些翻译后修饰。第二个具体目标是建立体外功能研究 PAX 3-FOXO 1融合蛋白与DNA和蛋白质辅助调节因子的相互作用。PAX 3-FOXO 1相互作用 将通过荧光检测结合试验和筛选机会靶来研究与DNA的结合 用于共结晶。反式激活结构域介导的蛋白质-蛋白质相互作用将使用 沉降速度分析超滤和核磁共振相结合 光谱，以确定在未来的研究中可能是药物的相互作用。这些目标将共同促进 PAX 3-FOXO 1融合蛋白与罕见病表型关系的探讨 腺泡状横纹肌肉瘤最终，通过这项试点研究开发的体外系统将被利用， 为了确定PAX 3-FOXO 1融合蛋白如何与伴侣相互作用， 为腺泡状横纹肌肉瘤的研究奠定了基础，并为下游高通量文库筛选奠定了基础。 能够破坏由PAX 3-FOXO 1反式激活结构域介导的相互作用的先导化合物。