Screening for Cys-Reactive Ligands to Target PAX3-FOXO1

补充申请U54 CA231630 PI：Angela Koehler，共同PI：Sara Buhrlage，Jarrod Marto 摘要 肺泡型横纹肌肉瘤(ARMS)是一种致死性的儿科癌症，由致癌因素引起 转录因子融合蛋白PAX3-FOXO1(P3F)。虽然遗传数据表明P3F是一种 潜在的治疗靶点，转录因子通常被认为是对药学的挑战 目标。本U54附录的目标是将药物识别策略扩展到我们的 目前的小分子微阵列(SMM)和实施共价配体筛选 P3F蛋白半胱氨酸残基，从而增加了潜在候选药物的池。要做到这一点 目标，团队将通力合作，追求三个目标。首先，克勒博士的团队将增加 生产纯化的全长P3F和截短形式的含有 Cys-793，一个被确定为辅因子CBP关键介体的残基。第二，克勒博士的团队 将与Buhrlage博士和Marto博士合作，优化全CyS反应的筛选条件 库使用池方法来增加吞吐量。将验证个别命中 通过LC-MS/MS确认生化结果，如共价修饰和 化学计量学。最后，将对共价配体的筛选进行二次检测，如 细胞热位移或报告分析，在SMM药物发现的母公司U45中概述。 克勒博士与布拉奇博士和马托博士之间的合作将扩展U54 团队的药物发现能力，并使更全面和有效的评估 靶向P3F的候选药物。在未来，这种技术方法可以扩展到 用于候选药物鉴定的其他转录因子。

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.