STRATEGIES TO TARGET HUMAN D-ASPARTATE OXIDASE AND MODULATE ENDOGENOUS D-ASPARTATE LEVELS IN SCHIZOPHRENIA
D-aspartate (D-Asp) is abundant in mammal brain at embryonal level while it rapidly decreases at post-natal stages due to the enhanced activity of D-aspartate oxidase (DASPO). D-Asp participates to glutamatergic neurotransmission and alterations of physiological metabolism are linked to pathologies. Depletion of D-Asp observed in post-mortem brain samples of schizophrenia patients has been correlated with excessive degradation by DASPO and causes the typical reduction of neurotransmission. D-Asp administration improves neurotransmission, synaptic plasticity and dendritic length, therefore the modulation of endogenous levels of D-Asp is interesting to develop potential therapies. To this aim, DASPO is a significant target in the absence of evidence for key regulators in the synthesis of D-Asp. 5-aminonicotinic acid (5-ANA) and olanzapine are mild inhibitors of human DASPO (hDASPO), thus new potential inhibitors were synthetised starting from the latter and their efficacy was tested in the Amplex UltraRed inhibition assay. While 5-ANA derivatives resulted inactive on hDASPO and were later repurposed on alternative targets, olanzapine derivatives showed interesting activity. Among a panel of 20 molecules, six paired the inhibition activity of olanzapine and X-ray crystallography was used to investigate molecular interactions and understand how to improve inhibition. The structure of wild type hDASPO was solved for the first time by molecular replacement and two out of six derivatives were modelled in the structures as non-competitive inhibitors in possible allosteric sites. Alongside, we would like to promote DASPO degradation via the proteasomes. To this aim, PROTAC molecules were synthetised linking olanzapine to either lenalidomide or (VH032)-Me designed to recruit Cereblon or Von Hippel–Lindau (VHL) E3 ligases, respectively. These compounds were tested both in vitro and in cell lines for the interaction with DASPO and E3 ligases, and for the formation of ternary complexes necessary for DASPO proteasomal degradation. Among them, PROTAC 1 showed ternary complex formation with a KD = 1.6 μM and induced hDASPO degradation in a neuroblastoma cell line after 48 hours of treatment. Ternary complex formation will be further investigated by NMR and SPR assays to enable complex isolation and facilitate future X-ray crystallography studies. In conclusion, on one side, inhibitor development and their structural characterisation can allow more specific modification of hit compounds to obtain potent molecules against hDASPO activity; on the other side, PROTAC technology was applied successfully for the first time to an enzymatic target and demonstrates the suitability of degradation strategy to target hDASPO. Both approaches open new avenues to regulate endogenous D-Asp levels, which may ultimately benefit patients by improving neurotransmission.
D-aspartate, hDASPO, neurotransmission, schizophrenia, inhibitor, degradation, ligase, crystallography, Settore BIOS-08/A - Biologia molecolare
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