Effect of ligand binding on human D‐amino acid oxidase: Implications for the development of new drugs for schizophrenia treatment

Abstract

In human brain the flavoprotein D-amino acid oxidase (hDAAO) is responsible for the degradation of the neuromodulator D-serine, an important effector of NMDA-receptor mediated neurotransmission. Experimental evidence supports the concept that D-serine concentration increase by hDAAO inhibition may represent a valuable therapeutic approach to improve the symptoms in schizophrenia patients. This study investigated the effects on hDAAO conformation and stability of the substrate D-serine (or of the pseudo-substrate trifluoro-D-alanine), the FAD cofactor, and two inhibitors (benzoate, a classical substrate-competitive inhibitor and the drug chlorpromazine (CPZ), which competes with the cofactor). We demonstrated that all these compounds do not alter the interaction of hDAAO with its physiological partner pLG72. The ligands used affect the tertiary structure of hDAAO differently: benzoate or trifluoro-D-alanine binding increases the amount of the holoenzyme form in solution and stabilizes the flavoprotein, while CPZ binding favors a protein conformation resembling that of the apoprotein, which is more sensitive to degradation. Interestingly, the apoprotein form of hDAAO binds the substrate D-serine: this interaction increases FAD binding thus increasing the amount of active holoenzyme in solution. Benzoate and CPZ similarly modify the short-term cellular D-serine concentration but affect the cellular concentration of hDAAO differently. In conclusion, the different alteration of hDAAO conformation and stability by the ligands used represents a further parameter to take into consideration during the development of new drugs to cope schizophrenia.

Structure and kinetic properties of humand‐aspartate oxidase, the enzyme‐controllingd‐aspartate levels in brain

“…55 The plot of the absorbance change at 501 nm as a function of the 5-An concentration showed a hyperbolic behavior with a K d = 10.2 ± 2.4 µM ( Figure 5A and Table 1), a value slightly higher than the K i value previously reported (K i = 3.80 ± 0.96 µM). 55 The plot of the absorbance change at 501 nm as a function of the 5-An concentration showed a hyperbolic behavior with a K d = 10.2 ± 2.4 µM ( Figure 5A and Table 1), a value slightly higher than the K i value previously reported (K i = 3.80 ± 0.96 µM).…”

“…Gel permeation chromatography indicates that the apoprotein form of hDASPO is present in solution as a 2:1 molar ratio between a monomeric (~39 kDa) and a trimeric species (100 ± 7 kDa). 55 The FAD dissociation constant (K d ) was determined from changes in hDASPO fluorescence intensity during titration of the apoprotein with FAD (Supplementary Figure 6): a K d of 33 ± 2.7 nM was estimated. Thermal stability of hDASPO is increased by the presence of the cofactor (the apoprotein form is slightly less stable than the holoenzyme, which is further stabilized by an excess of free FAD) or by a saturating 5-An concentration ( Table 1, Supplementary Figure 5).…”

Triazole–Dithiocarbamate Based Selective Lysine Specific Demethylase 1 (LSD1) Inactivators Inhibit Gastric Cancer Cell Growth, Invasion, and Migration

“…60 Chlorpromazine (CPZ), a dopamine D2 receptor antagonist introduced in the treatment of schizophrenia in the early 1950s, acts as a FAD-competitive inhibitor of human D-amino acid oxidase (hDAAO) with similar structure. 61 K001 that competes for FAD binding, inhibits cryptochrome (CRY) ubiquitylation and lengthens the circadian cycle. 62 There are some other papers published on FAD ejectors.…”

The degradation (by distinct pathways) of human d‐amino acid oxidase and its interacting partner pLG72 – two key proteins in d‐serine catabolism in the brain

“…Because the expression level in the reference cell line is below the detection limit even when the degradation pathways are blocked, cell clones overexpressing the target proteins were used. Of note, the addition of an EGFP tag (or its variants with modified fluorescence) to hDAAO was shown not to affect its biochemical and binding properties and subcellular localization .…”