Screening for Cys-Reactive Ligands to Target PAX3-FOXO1

筛选针对 PAX3-FOXO1 的 Cys 反应性配体

基本信息

批准号：
10611002
负责人：
CHRISTOPHER M COUNTER
金额：
$ 36.94万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-01 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10611002
关键词：
Alveolar Rhabdomyosarcoma Binding Biochemical Biological Assay Biology Cell Line Cells Chemicals Chimeric Proteins Chromosomal translocation Collaborations Critical Pathways DNA Dana-Farber Cancer Institute Data Development Disease Drug Targeting Epitopes FOXO1A gene Family member Fire - disasters Future Genetic Genetic Transcription Goals Grant Individual Laboratories Length Libraries Ligands Luciferases Malignant Childhood Neoplasm Mass Spectrum Analysis Mediator of activation protein Modification Monitor Oncogenic Oxidation-Reduction PAX3 gene Palate Parents Patient-Focused Outcomes Pharmaceutical Preparations Production Proteins Reporter Scientist Secondary to Site Structure Transactivation Transcript Translational Research Trypsin Validation Work base candidate identification childhood sarcoma cofactor design drug candidate drug discovery experience genetic approach innovation member multidisciplinary mutant novel therapeutics protein complex screening sensor small molecule stoichiometry therapeutic target transcription factor

项目摘要

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.

补充申请 U54 CA231630 PI：Angela Koehler，Co-PI：Sara Buhrlage、Jarrod Marto 抽象的腺泡状横纹肌肉瘤 (ARMS) 是一种致命的儿科癌症，由致癌物质驱动转录因子融合蛋白 PAX3-FOXO1 (P3F) 而遗传数据表明 P3F 是一种转录因子融合蛋白。潜在的治疗靶点，转录因子通常被认为具有挑战性的药理学该 U54 补充剂的目标是将药物识别策略扩展到我们之外。当前的小分子微阵列（SMM）并针对其实施共价配体筛选 P3F 蛋白的 Cys 残基，从而增加了潜在候选药物的库以实现这一目标。目标，团队将合作实现三个目标：首先，Koehler 博士的团队将增加。纯化的全长 P3F 以及包含的蛋白质的截短形式的生产 Cys-793，一种被确定为辅助因子 CBP 的关键介质的残留物其次，Koehler 博士的团队。将与 Buhrlage 和 Marto 博士合作，优化完全 cys 反应性的筛选条件利用池化方法来提高吞吐量的库将得到验证。通过 LC-MS/MS 确认生化结果，例如共价修饰和最后，共价配体筛选命中将进行二次测定，例如细胞热位移或报告基因检测，在用于 SMM 药物发现的母版 U45 中概述。 Koehler 博士、Buhrlage 博士和 Marto 博士之间的合作将扩展 U54 团队的药物发现能力，并能够更彻底、更有效地评估未来，这种技术方法可以扩展到靶向 P3F 的候选药物。用于候选药物鉴定的其他转录因子。