PROTAC-mediated degradation of peroxisomal d-aspartate oxidase: A novel strategy to modulate d-aspartate homeostasis for schizophrenia treatment

D-Amino acids, once considered negligible in mammalian physiology, are now recognized as key neuromodulators. Among them, D-aspartate (D-Asp) regulates NMDAR and mGlu5 receptor function, influencing neurodevelopment, synaptic plasticity and cognition. In humans, postnatal brain D-Asp levels decline due to the upregulation of D-aspartate oxidase (hDASPO), the peroxisomal flavoenzyme responsible for D-Asp catabolism. Altered D-Asp homeostasis, characterized by reduced D-Asp and elevated hDASPO expression, has been associated with schizophrenia, supporting therapeutic strategies aimed at restoring physiological D-Asp levels. Although several small-molecule inhibitors of hDASPO have been reported, no potent or clinically viable candidates have emerged. Here, we investigated an alternative strategy based on targeted protein degradation to modulate hDASPO abundance. Guided by STD-NMR, we designed a series of heterobifunctional degraders integrating a hDASPO-binding ligand (olanzapine) with established E3 ligase recruiters (lenalidomide or VH032–Me) and aliphatic linkers of varying lengths. The resulting bifunctional compounds were synthesized and evaluated in biochemical assays for their ability to bind hDASPO. Most compounds retained micromolar inhibitory activity, indicating that derivatization at olanzapine position 10 preserves target engagement, with CRBN-recruiting analogues generally outperforming their VHL-based counterparts. Selected compounds were further investigated in cellular models, where PROTAC 17 demonstrated robust target engagement, positive cooperativity in ternary complex formation and dose-dependent degradation of hDASPO. Together, these findings establish the first proof-of-concept for hDASPO degradation via the PROTAC approach and provide a foundation for therapeutic strategies aimed at re-establishing D-Asp homeostasis in neuropsychiatric disorders characterized by NMDAR dysregulation.