Identification of Novel d-Amino Acid Oxidase Inhibitors by in Silico Screening and Their Functional Characterization in Vitro

Abstract

D-amino acid oxidase (DAO) is a degradative enzyme that is stereospecific for D-amino acids, including D-serine and D-alanine, which are potential coagonists of the N-methyl-D-aspartate (NMDA) receptor. Dysfunction of NMDA receptor-mediated neurotransmission has been implicated in the onset of various mental disorders such as schizophrenia. Hence, a DAO inhibitor that augments the brain levels of D-serine and/or D-alanine and thereby activates NMDA receptor function is expected to be an antipsychotic drug, for instance, in the treatment of schizophrenia. In the search for potent DAO inhibitor(s), a large number of compounds were screened in silico, and several compounds were estimated as candidates. These compounds were then characterized and evaluated as novel DAO inhibitors in vitro. The results reported in this study indicate that some of these compounds are possible lead compounds for the development of a clinically useful DAO inhibitor and have the potential to serve as active site probes to elucidate the structure-function relationships of DAO.

Biosynthesis of d-aspartate in mammals: the rat and human homologs of mouse aspartate racemase are not responsible for the biosynthesis of d-aspartate

“…The following day, the medium was replaced with 1.2 ml of fresh medium and the cells were cultured for a further 0, 1 or 2 days before extraction of amino acids from the cells and culture media. The amino acids in the cells and culture media were extracted using methanol, as described previously (Katane et al 2013;Long et al 1998) with some modifications. Specifically, the culture media were recovered and centrifuged at 300×g for 5 min at 4 °C to remove the cell debris.…”

Characterization of the Enzymatic and Structural Properties of Human D-Aspartate Oxidase and Comparison with Those of the Rat and Mouse Enzymes

“…Crude extracts were prepared from cells transformed with pRSET-HishDDO and pRSET-His-mDASPO, as described previously. 33,36) For cells transformed with pRSET-His-rDDO, the culture was grown to A 620 =0.5 and then incubated for an additional 30 min at a reduced temperature (26°C). After the addition of 0.5 mM isopropyl-β-D-thiogalactopyranoside, the culture was incubated at 26°C for a further 16 h. The cells were then centrifuged at 10000×g for 10 min at 4°C, and crude extract was prepared using BugBuster Protein Extraction Reagent and Lysonase Bioprocessing Reagent (Novagen) in the presence of 50 µM FAD and protease inhibitors (Nacalai Tesque), according to the manufacturer's instructions.…”

“…To examine the effects of various compounds on the activity of DDO, each compound was added to the reaction mixture individually and its relative inhibitory activity was normalized to that of the enzyme alone. The IC 50 values of the compounds tested were determined using the following formula, as described previously 33) : IC 50 =10…”

“…16,17) Overall, human DDO is an attractive therapeutic target and several compounds that inhibit the activity of mammalian DDO in vitro have been identified to date; however, the inhibitory potency of these compounds is limited. [28][29][30][31][32][33] Preclinical studies using experimental rodents are essential prerequisites for evaluating the in vivo effects of clinically useful enzyme inhibitors. However, species-related differences in some properties of target enzymes occur frequently and can lead to inaccurate evaluation of the in vivo effects of the inhibitors; therefore, it is vital to characterize and compare the properties of human enzymes with those of their rodent homologs.…”

A sensitive assay for measuring aspartate-specific amino acid racemase activity

“…Crude extract was prepared from cells transformed with pRSET-HishDDO, as described previously [31]. The specific activity of the DDO extract toward 20 mM d-Asp was 0.197 mol/min/mg, which was determined using a colorimetric assay for 2-oxo acid production, as described previously [32].…”