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

Hopkins, Seth C.; Heffernan, Michelle L R; Saraswat, Lakshmi D.; Bowen, Carrie A.; Melnick, Laurence; Hardy, Larry W.; Orsini, Michael A.; Allen, Michael S.; Koch, Patrick; Spear, Kerry L.; Foglesong, Robert J.; Soukri, Mustapha; Chytil, Milan; Fang, Q. Kevin; Jones, Sarah R.; Varney, Mark A.; Panatier, Aude; Oliet, Stéphane H. R.; Pollegioni, Loredano; Piubelli, Luciano; Molla, Gianluca; Nardini, M.; Large, Thomas H.

doi:10.1021/jm4002583

Cited by 32 publications

(44 citation statements)

References 61 publications

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“…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.…”

Section: Discussionmentioning

confidence: 99%

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

Takahashi

Furukawara

Omae

et al. 2014

Appl Environ Microbiol

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D-Amino acid oxidase (DAO) is a biotechnologically attractive enzyme that can be used in a variety of applications, but its utility is limited by its relatively poor stability. A search of a bacterial genome database revealed a gene encoding a protein homologous to DAO in the thermophilic bacterium Rubrobacter xylanophilus (RxDAO). The recombinant protein expressed in Escherichia coli was a monomeric protein containing noncovalently bound flavin adenine dinucleotide as a cofactor. This protein exhibited oxidase activity against neutral and basic D-amino acids and was significantly inhibited by a DAO inhibitor, benzoate, but not by any of the tested D-aspartate oxidase (DDO) inhibitors, thus indicating that the protein is DAO. RxDAO exhibited higher activities and affinities toward branched-chain D-amino acids, with the highest specific activity toward D-valine and catalytic efficiency (k cat /K m ) toward D-leucine. Substrate inhibition was observed in the case of D-tyrosine. The enzyme had an optimum pH range and temperature of pH 7.5 to 10 and 65°C, respectively, and was stable between pH 5.0 and pH 8.0, with a T 50 (the temperature at which 50% of the initial enzymatic activity is lost) of 64°C. No loss of enzyme activity was observed after a 1-week incubation period at 30°C. This enzyme was markedly inactivated by phenylmethylsulfonyl fluoride but not by thiol-modifying reagents and diethyl pyrocarbonate, which are known to inhibit certain DAOs. These results demonstrated that RxDAO is a highly stable DAO and suggested that this enzyme may be valuable for practical applications, such as the determination and quantification of branched-chain D-amino acids, and as a scaffold to generate a novel DAO via protein engineering.T he flavoenzyme D-amino acid oxidase (DAO; EC 1.4.3.3) catalyzes the oxidative deamination of neutral and basic D-amino acids but not of acidic D-amino acids, which are substrates of D-aspartate oxidase (DDO). DAO was first identified in pig kidney (pkDAO) by Krebs in 1935 (1) and subsequently has been found mainly in eukaryotic organisms, ranging from fungi to humans (2). These eukaryotic DAOs have been extensively studied as a model of the oxidase-dehydrogenase class of flavoproteins. In fungi, the enzyme plays a role in the assimilation of D-amino acids for cell growth and also in the detoxification of D-amino acid toxicity (2, 3). In vertebrates, this enzyme is mainly localized in the liver and kidney, and it metabolizes both exogenous and endogenous D-amino acids (2). In addition, the enzyme plays a specific role in the regulation of D-serine, a coagonist for the N-methyl-Daspartate receptor, in the brain (2).In contrast, for a long time, it was thought that DAO did not exist in prokaryotic organisms. However, this enzyme was recently identified in Arthrobacter protophormiae (ApDAO) and Streptomyces coelicolor (ScDAO) (4, 5). In addition to these bacterial species, a search of a prokaryotic genome database revealed a wide distribution of DAO homologous proteins in bacteria, especially a...

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Section: Discussionmentioning

confidence: 99%

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

Takahashi

Furukawara

Omae

et al. 2014

Appl Environ Microbiol

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“…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.…”

Section: Introductionmentioning

confidence: 99%

“…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].…”

Section: Introductionmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Terry-Lorenzo

Chun²,

Brown

et al. 2014

Bioscience Reports

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The NMDAR (N-methyl-D-aspartate receptor) is a central regulator of synaptic plasticity and learning and memory. hDAAO (human D-amino acid oxidase) indirectly reduces NMDAR activity by degrading the NMDAR co-agonist D-serine. Since NMDAR hypofunction is thought to be a foundational defect in schizophrenia, hDAAO inhibitors have potential as treatments for schizophrenia and other nervous system disorders. Here, we sought to identify novel chemicals that inhibit hDAAO activity. We used computational tools to design a focused, purchasable library of compounds. After screening this library for hDAAO inhibition, we identified the structurally novel compound, ‘compound 2’ [3-(7-hydroxy-2-oxo-4-phenyl-2H-chromen-6-yl)propanoic acid], which displayed low nM hDAAO inhibitory potency (Ki=7 nM). Although the library was expected to enrich for compounds that were competitive for both D-serine and FAD, compound 2 actually was FAD uncompetitive, much like canonical hDAAO inhibitors such as benzoic acid. Compound 2 and an analog were independently co-crystalized with hDAAO. These compounds stabilized a novel conformation of hDAAO in which the active-site lid was in an open position. These results confirm previous hypotheses regarding active-site lid flexibility of mammalian D-amino acid oxidases and could assist in the design of the next generation of hDAAO inhibitors.

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“…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.…”

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confidence: 99%

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

Hin

Duvall

Berry

et al. 2016

Bioorganic & Medicinal Chemistry Letters

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A series of 3-substituted 5-hydroxy-1,2,4-triazin-6(1H)-one derivatives were designed and synthesized as a new class of D-amino acid oxidase (DAAO) inhibitors. Some of the newly synthesized derivatives showed potent inhibitory activity against human DAAO with IC50 values in the nanomolar range. Among them, 6-hydroxy-3-phenethyl-1,2,4-triazin-5(2H)-one 6b and 3-((6-fluoronaphthalen-2-yl)methylthio)-6-hydroxy-1,2,4-triazin-5(2H)-one 6m were found to be metabolically stable in mouse liver microsomes. In addition, compound 6b was found to be orally available in mice and able to enhance plasma D-serine levels following its co-administration with D-serine compared to the oral administration of D-serine alone.

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

Cited by 32 publications

References 61 publications

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

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

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

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

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