针对大型内在无序蛋白的非共价小分子探针发现

Fusion-positive alveolar rhabdomyosarcoma (ARMS), is a rare pediatric cancer often characterized by the pathognomonic fusion protein PAX3::FOXO1. Similar to other highly disordered transcription factors, PAX3::FOXO1 lacks typical small-molecule binding pockets thus making drug development difficult by conventional structural oriented design or traditional screening. Currently, there is no direct therapeutic approach to treat children with fusion-positive ARMS and patients are treated with ineffective traditional chemotherapeutic agents. Previously, we screened a library of small molecules against PAX3::FOXO1 via Small-Molecule Microarrays (SMMs) and identified a compound that shows selective target engagement in thermal shift assay but no apparent cellular activity. Furthermore, we are exploring targeted degradation with PROTACs due to the slow turn-over rate of this target. We have identified a scalable protein purification strategy to achieve stable, functional recombinant PAX3::FOXO1 and we are in the process of expanding the SMMs libraries up to 300,000 compounds. To validate hits from those high throughput screenings, we developed lentiviral transduced ARMS cell lines expressing HiBiT-tagged PAX3::FOXO1, enabling plate-based reporter assays as well as thermal shift assays. Small molecules are highly advantageous in their administration, delivery, and patient accessibility. We hope to address some of the unmet needs in the space of small molecule drug discovery against intrinsically disordered oncoprotein and transcription factors.