Screening for Cys-Reactive Ligands to Target PAX3-FOXO1

补充申请U 54 CA 231630 PI：Angela Koehler，Co-PI：Sara Buhrlage，Jarrod Marto 摘要 腺泡状横纹肌肉瘤（ARMS）是一种致命的儿科癌症， 转录因子融合蛋白PAX 3-FOXO 1（P3 F）。虽然遗传数据表明P3 F是一种 潜在的治疗靶点，转录因子通常被认为是具有挑战性的药理学 目标的这U 54补充的目标是扩大药物识别策略， 目前的小分子微阵列（SMM），并实施共价配体筛选， P3 F蛋白Cys残基，从而增加潜在候选药物的库。实现这一 目标，团队将合作追求三个目标。首先，克勒博士的团队会增加 纯化的全长P3 F以及截短形式的蛋白质的生产，所述截短形式的蛋白质含有 Cys-793，一种被鉴定为辅助因子CBP的关键介体的残基。第二，克勒博士的团队 我将与Buhrlage博士和Marto博士合作，优化筛选条件，以获得完整的cys反应性 库利用池化方法来增加吞吐量。将对单个命中进行验证 通过LC-MS/MS确认生化结果，如共价修饰和 化学计量最后，共价配体筛选命中物将经受二次测定，例如 细胞热位移或报告基因测定，在用于SMM药物发现的母公司U45中概述。 Koehler博士、Buhrlage博士和Marto博士之间的合作将扩大U 54 团队的药物发现能力，并能够更彻底和有效地评估 靶向P3 F的候选药物。在未来，这种技术方法可以扩展到 用于药物候选物鉴定的其它转录因子。

Supplemental application U54 CA231630 PI: Angela Koehler, Co-PIs: Sara Buhrlage, Jarrod Marto Abstract Alveolar rhabdomyosarcoma (ARMS) is a lethal pediatric cancer driven by the oncogenic transcription factor fusion protein PAX3-FOXO1 (P3F). While genetic data demonstrate P3F is a potential therapeutic target, transcription factors are often considered challenging pharmalogical targets. The goal of this U54 supplement is to expand drug identification strategies past our current small-molecule microarrays (SMMs) and implement covalent ligand screening against P3F protein Cys residues, thus increasing the pool of potential drug candidates. To achieve this goal, team will collaborate to pursue three objectives. First, Dr. Koehler’s team will increase production of purified full-length P3F as well as truncated forms of the protein which contain Cys-793, a residue identified as a critical mediator of cofactor CBP. Second, Dr. Koehler’s team will work with Drs. Buhrlage and Marto to optimize screening conditions for a full cys-reactive library utilizing a pooling-approach to increase throughput. Individual hits will be validated through LC-MS/MS to confirm biochemical results, such as covalent modifications and stoichiometry. Lastly, covalent ligand screen hits will be subjected to secondary assays, such as cellular thermal shift or reporter assays, outlined in the parent U45 for SMM drug discovery. This collaboration between Dr. Koehler and Drs. Buhrlage and Marto will expand the U54 team’s drug discovery capabilities, and enable a more thorough and efficient assessment of candidate drugs to target P3F. In the future, this technological approach could be extended to other transcription factors for drug candidate identification.