Discovery of a CNS active GSK3 degrader using orthogonally reactive linker screening

Bifunctional targeted protein degraders, also known as Proteolysis Targeting Chimeras (PROTACs), are an emerging drug modality that may offer a new approach to understand and treat neurodegenerative diseases. These molecules have a non-occupancy based, sub-stoichiometric mechanism of action which results in removal of target proteins from cells, presenting new opportunities to interrogate and intervene in pathogenic signalling. Identifying chemical starting points for PROTACs remains a largely empirical process and the design rules for identifying in vivo quality tools to interrogate new therapeutic hypotheses in the Central Nervous System (CNS) have yet to be established. We demonstrate a concept of using orthogonally reactive linker reagents, that allowed us to construct screening libraries varying the E3 ligase recruiting ligand, the target protein recruiting ligand and the linker simultaneously and test compounds directly in cells. This led us identify a molecule directly from our screen that upon further profiling demonstrated rapid and selective Glycogen Synthase Kinase 3 (GSK3) degradation in cells, as well as CNS in vivo activity in mouse. Our findings provide new opportunities to investigate the role of GSK3 paralogs in cellular and in vivo disease models and demonstrate a successful Direct-to-Biology approach, with broad potential for identifying in vivo quality bifunctional chemical probes for CNS disease concepts.