PROTAC technology for the degradation of hDASPO: from design to synthesis to biophysical and cellular evaluation

D-Aspartate (D-Asp) is a D-amino acid able to stimulate postsynaptic N-Me-D-Asp (NMDA) receptors in the central nervous system, affecting synaptic transmission, plasticity and cognition. The catabolism of D-Asp is due to the enzyme D-Aspartate Oxidase (DASPO): patients affected by schizophrenia present a low concentration of D-Asp in the brain due to the overexpression of DASPO 1, and the dietary assumption of additional D-Asp has been found to be beneficial. 2 For this reason, it was hypothesized that the modulation of human DASPO (hDASPO) activity could be an alternative therapeutic approach for the treatment of this psychiatric disease. Instead of classical inhibition, the modulation of hDASPO activity was attempted through degradation of the enzyme itself exploiting Proteolysis Targeting Chimeras (PROTACs). These compounds are made of a ligand of the protein of interest (POI) and a ligand of an E3 ligase connected by a linker. After formation of the ternary complex POI – PROTAC – E3 ligase, the POI is polyubiquitinated and degraded, with entrance of the PROTAC in a new degradation cycle. Olanzapine, a common second-generation antipsychotic drug, was found to present a good binding affinity and inhibition activity of DASPO. 3 Considering also the known tolerability and side effects of the drug, olanzapine was chosen as POI ligand. Lenalidomide and (VH-032)-Me were selected as recruiters of the E3 ligases Cereblon and Von Hippel Lindau, respectively. The two moieties were connected with flexible aliphatic linkers presenting a 1,4-disubstituted 1,2,3-triazole, a motif which can be easily introduced with a copper catalyzed azide alkyne cycloaddition (CuAAC), giving the final molecules with high purity and increasing the molecule’s stability and solubility. The binding affinity of the synthetized PROTACs was tested through in vitro enzyme assays and MST, confirming the ability of most of the synthetized PROTACs to bind to both hDASPO or the respective E3 ligase. Also, the formation of the ternary complex was confirmed via MST and is being studied via DOSY-NMR. Finally, the degradation activity of the synthetized PROTACs was evaluated on two different cell lines, leading to one optimal candidate. References: [1] Errico et al., 2018, Front. Psychiatry, 9. [2] Errico et al., 2013, Psychiatr. Res., 47(10), 1432–1437 [3] Sacchi et al., 2017, Sci. Rep., 7(1)