DEVELOPMENT OF AN ARRAY OF SECONDARY ASSAYS FOR THE SELECTION AND VALIDATION OF TRUE-POSITIVE HITS IDENTIFIED IN SCREENING CAMPAIGNS ON MOLECULAR TARGETS OF THERAPEUTIC INTEREST

To enhance the reliability of High-Throughput Screening (HTS), particularly in campaigns targeting TET (Ten-eleven translocation) methylcytosine dioxygenase family, this thesis focuses on developing a robust secondary assay strategy. Ten-Eleven Translocation (TET) proteins are crucial dioxygenase involved in DNA demethylation, a fundamental epigenetic process. Mutations in TET proteins are frequently observed in hematologic malignancies, where such proteins often act as tumor suppressors. However, their role is complex, with some studies suggesting tumor-promoting functions in specific contexts, making it a challenging yet promising drug target. Therefore, a comprehensive HTS campaign targeting TET proteins must consider identifying both activators and inhibitors, as either modulation could be therapeutically beneficial depending on the disease context1. This thesis introduces a comprehensive strategy to enhance the reliability of HTS by mitigating the impact of interfering compounds, non-target directed compounds, and assay-related limitations, thereby facilitating the discovery of true TET modulators. Moreover, the impact of this project is anticipated to receive direct and prompt implementation and validation by integrating the developed secondary assays into drug discovery projects performed at Axxam SpA as part of the research activities conducted in collaboration with international academic and industrial key players in the pharmaceutical field.