Design, synthesis and biological evaluation of PROTACs targeting hDASPO

Design, synthesis and biological evaluation of PROTACs targeting hDASPO • Background Even though D-amino acids were considered to have negligible functions in biological processes, today we know that they play significant and specific roles in the organism.[1] In particular, D-Aspartate (D-Asp) is involved in the central nervous system, as it stimulates postsynaptic N-Me-D-Asp (NMDA) receptors, affecting synaptic transmission, plasticity and cognition, and is catabolized by the enzyme D-Aspartate Oxidase (DASPO). Interestingly, patients affected by schizophrenia present a low concentration of D-Asp in the brain due to the overexpression of DASPO[2], and the dietary assumption of additional D-Asp has been found to be beneficial.[3] 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. • Methods Instead of classical inhibition, the modulation of hDASPO activity may be 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. • Results Olanzapine, a common second-generation antipsychotic drug, was found to present a good binding affinity and inhibition activity of DASPO. [4] 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, and the two moieties were connected with flexible aliphatic linkers presenting a 1,4-disubstituted 1,2,3-triazole, a motif which increases the molecule’s stability and solubility and can create H – bonds. STD-NMR studies[5] on Olanzapine allowed to select N10 as functionalization point, due to its little interactions with the enzyme, together with the good reactivity. An alkyne functionality was therefore introduced on N10 of Olanzapine. Azido carboxylic acids were obtained from bromo carboxylic acids and were reacted with the amino group of the respective E3 ligase recruiter through an amide coupling. Eventually, a copper catalyzed azide alkyne cycloaddition (CuAAC) allowed to connect the two moieties, giving products with purity > 95%, confirmed by HPLC. The binding affinity of 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. The formation of the ternary complex was studied and confirmed through MST. • Conclusions In conclusion, two libraries of PROTACs were designed and synthetized. Their binding affinity was evaluated and the ternary complex formation was confirmed through MST. NMR experiments to further confirm the ternary complex formation and cellular assays to evaluate the degradation activity are currently ongoing. [1] Errico et al., 2012, Amino Acids, 43(5), 1861–1871. [2] Errico et al., 2018, Front. Psychiatry, 9. [3] Errico et al., 2013, Psychiatr. Res., 47(10), 1432–1437 [4] Sacchi et al., 2017, Sci. Rep., 7(1) Wagstaff et al., 2013, Mol. BioSyst., 9(4), 571–577