Structural, Kinetic, and Pharmacodynamic Mechanisms of d-Amino Acid Oxidase Inhibition by Small Molecules

Abstract

We characterized the mechanism and pharmacodynamics of five structurally distinct inhibitors of d-amino acid oxidase. All inhibitors bound the oxidized form of human enzyme with affinity slightly higher than that of benzoate (Kd ≈ 2-4 μM). Stopped-flow experiments showed that pyrrole-based inhibitors possessed high affinity (Kd ≈ 100-200 nM) and slow release kinetics (k < 0.01 s(-1)) in the presence of substrate, while inhibitors with pendent aromatic groups altered conformations of the active site lid, as evidenced by X-ray crystallography, and showed slower kinetics of association. Rigid bioisosteres of benzoic acid induced a closed-lid conformation, had slower release in the presence of substrate, and were more potent than benzoate. Steady-state d-serine concentrations were described in a PK/PD model, and competition for d-serine sites on NMDA receptors was demonstrated in vivo. DAAO inhibition increased the spatiotemporal influence of glial-derived d-serine, suggesting localized effects on neuronal circuits where DAAO can exert a neuromodulatory role.

A Highly Stable d -Amino Acid Oxidase of the Thermophilic Bacterium Rubrobacter xylanophilus

“…Although the production of branched-chain D-amino acids remains unknown in R. xylanophilus, the substrate specificity indicates the possibility that RxDAO might be involved in the functions of branched-chain D-amino acids through their degradation. In pkDAO, a flexible loop near the active site, called the active-site lid that opens and closes for substrate entry and product exit, is involved in substrate specificity (33,34). In particular, the flexibility of Y224 in the lid is likely important for its broad substrate specificity.…”

Novel humanD-amino acid oxidase inhibitors stabilize an active-site lid-open conformation

“…This link between D -serine and NMDAR function suggested that, by controlling D -serine brain levels, DAAO and serine racemase, the D -serine-synthesizing enzyme [11], are also involved in regulating the key brain functions. Despite the apparent low levels of DAAO in forebrain regions [12], pharmacological studies with DAAO inhibitors [13,14], genetic studies in DAAO knockout animals [15,16] and genetic studies with serine racemase-mutated mice [17,18] have all clearly indicated that D -serine regulatory enzymes impact NMDAR function, synaptic function and cognitive ability. Furthermore, chemical regulators of D -serine metabolism could be effective disease treatments.…”

“…Furthermore, chemical regulators of D -serine metabolism could be effective disease treatments. More specifically, as NMDAR hypofunction is a core pathway deficit in schizophrenia [19,20], DAAO inhibitors, perhaps in combination with D -serine systemic administration [21], have the potential to be effective treatments of schizophrenia via their capacity to increase D -serine in the brain and enhance NMDAR-dependent functions [13,14,22]. Moreover, although the mechanistic underpinnings of DAAO's roles in nociception are not fully understood, DAAO inhibitors have been shown to reduce pain in various rodent models of neuropathic pain [23–25].…”

“…Further efforts revealed that bioisosteric replacement of the acid could produce compounds which were also hDAAO inhibitors with low nM potency [22]. Guided by the knowledge that the active site could accommodate D -amino acids with large aromatic side chains, such as D -tryptophan [28] and D -DOPA [29], a newer generation of hDAAO inhibitors were designed that occupy the so-called ‘subpocket’ of the hDAAO active site [13,30,31]. Additional plasticity in the hDAAO active site beyond the re side of flavin and the subpocket has yet to be explored.…”

d-Amino acid oxidase inhibitors based on the 5-hydroxy-1,2,4-triazin-6(1H)-one scaffold

“…6 2-Substituted 6-hydroxy-1,2,4-triazine-3,5(2 H ,4 H )-diones such as 3 exhibited not only potent DAAO inhibitory activity but also improved metabolic stability compared to 1–2 in liver microsomes. 7 This secondary binding site was also exploited by carboxylate-based DAAO inhibitors such as 4 8 and 5 . 9 In the search for a new class of DAAO inhibitors, we have examined a variety of scaffolds that may serve as a bioisostere for the carboxylic acid moiety interacting with the active site of DAAO.…”