Limits...
Novel human D-amino acid oxidase inhibitors stabilize an active-site lid-open conformation.

Terry-Lorenzo RT, Chun LE, Brown SP, Heffernan ML, Fang QK, Orsini MA, Pollegioni L, Hardy LW, Spear KL, Large TH - Biosci. Rep. (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: *Discovery Research Department, Sunovion Pharmaceuticals, Marlborough, MA 01752, U.S.A.

ABSTRACT
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.

Show MeSH

Related in: MedlinePlus

Cpd 2 interacts with the active site of hDAAOIn this figure and in subsequent crystallography images, illustrated amino acid side chain carbons are uncoloured, carbons of FAD are yellow, and ligand carbons are differentially coloured. Compound structures of ligands included in the crystallographic images are displayed at the bottom of the images. (A) In the X-ray crystal structure, cpd 2 (green) binds in the active site of hDAAO adjacent to the FAD cofactor. The carboxylic acid moiety of cpd 2 forms four different hydrogen bonds (dashed lines) with Arg283, Tyr55 and Tyr228 of hDAAO. The aromatic ring of the Tyr55 side chain also forms a π–π stacking interaction with the coumarin of cpd 2 (π–π stacking interactions not shown for image clarity). (B) Unlike other hDAAO inhibitors described in the literature, cpd 2 forms hydrogen bonds with the carbonyl backbone of hDAAO Gln53 and forms a hydrogen bond with a water molecule (red sphere).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4127593&req=5

Figure 3: Cpd 2 interacts with the active site of hDAAOIn this figure and in subsequent crystallography images, illustrated amino acid side chain carbons are uncoloured, carbons of FAD are yellow, and ligand carbons are differentially coloured. Compound structures of ligands included in the crystallographic images are displayed at the bottom of the images. (A) In the X-ray crystal structure, cpd 2 (green) binds in the active site of hDAAO adjacent to the FAD cofactor. The carboxylic acid moiety of cpd 2 forms four different hydrogen bonds (dashed lines) with Arg283, Tyr55 and Tyr228 of hDAAO. The aromatic ring of the Tyr55 side chain also forms a π–π stacking interaction with the coumarin of cpd 2 (π–π stacking interactions not shown for image clarity). (B) Unlike other hDAAO inhibitors described in the literature, cpd 2 forms hydrogen bonds with the carbonyl backbone of hDAAO Gln53 and forms a hydrogen bond with a water molecule (red sphere).

Mentions: The binding mode of compound 2 to the hDAAO:FAD holoenzyme was determined by X-ray crystallography (crystallographic data in Table 2). In agreement with our kinetic observations (Figure 2), compound 2 occupied the active site of hDAAO adjacent to the FAD cofactor. The binding interaction was consistent with previously published cocrystal structures of hDAAO with small aryl carboxylic acid inhibitors or carboxylic acid bioisosteres [13,22,27,30] and included a bidentate hydrogen bond from the compound propanoate arm to the guanidinium group of Arg283 (Figure 3A). This propanoic acid-binding interaction was further stabilized by hydrogen bonds with Tyr228 and Tyr55 (Figure 3A). The Tyr55 interaction was only available following local rearrangement of the side chains of the active site because of inhibitor binding. This interaction with Tyr55 was not seen in past hDAAO–ligand cocrystal structures (for example, see Figure 4B). Additional interactions not observed in previous hDAAO–ligand structures included a hydrogen bond between the coumarin hydroxyl and the carbonyl backbone of Gln53 and a water molecule interaction with the cyclic oxygen and carbonyl of compound 2's coumarin ring (Figure 3B).


Novel human D-amino acid oxidase inhibitors stabilize an active-site lid-open conformation.

Terry-Lorenzo RT, Chun LE, Brown SP, Heffernan ML, Fang QK, Orsini MA, Pollegioni L, Hardy LW, Spear KL, Large TH - Biosci. Rep. (2014)

Cpd 2 interacts with the active site of hDAAOIn this figure and in subsequent crystallography images, illustrated amino acid side chain carbons are uncoloured, carbons of FAD are yellow, and ligand carbons are differentially coloured. Compound structures of ligands included in the crystallographic images are displayed at the bottom of the images. (A) In the X-ray crystal structure, cpd 2 (green) binds in the active site of hDAAO adjacent to the FAD cofactor. The carboxylic acid moiety of cpd 2 forms four different hydrogen bonds (dashed lines) with Arg283, Tyr55 and Tyr228 of hDAAO. The aromatic ring of the Tyr55 side chain also forms a π–π stacking interaction with the coumarin of cpd 2 (π–π stacking interactions not shown for image clarity). (B) Unlike other hDAAO inhibitors described in the literature, cpd 2 forms hydrogen bonds with the carbonyl backbone of hDAAO Gln53 and forms a hydrogen bond with a water molecule (red sphere).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4127593&req=5

Figure 3: Cpd 2 interacts with the active site of hDAAOIn this figure and in subsequent crystallography images, illustrated amino acid side chain carbons are uncoloured, carbons of FAD are yellow, and ligand carbons are differentially coloured. Compound structures of ligands included in the crystallographic images are displayed at the bottom of the images. (A) In the X-ray crystal structure, cpd 2 (green) binds in the active site of hDAAO adjacent to the FAD cofactor. The carboxylic acid moiety of cpd 2 forms four different hydrogen bonds (dashed lines) with Arg283, Tyr55 and Tyr228 of hDAAO. The aromatic ring of the Tyr55 side chain also forms a π–π stacking interaction with the coumarin of cpd 2 (π–π stacking interactions not shown for image clarity). (B) Unlike other hDAAO inhibitors described in the literature, cpd 2 forms hydrogen bonds with the carbonyl backbone of hDAAO Gln53 and forms a hydrogen bond with a water molecule (red sphere).
Mentions: The binding mode of compound 2 to the hDAAO:FAD holoenzyme was determined by X-ray crystallography (crystallographic data in Table 2). In agreement with our kinetic observations (Figure 2), compound 2 occupied the active site of hDAAO adjacent to the FAD cofactor. The binding interaction was consistent with previously published cocrystal structures of hDAAO with small aryl carboxylic acid inhibitors or carboxylic acid bioisosteres [13,22,27,30] and included a bidentate hydrogen bond from the compound propanoate arm to the guanidinium group of Arg283 (Figure 3A). This propanoic acid-binding interaction was further stabilized by hydrogen bonds with Tyr228 and Tyr55 (Figure 3A). The Tyr55 interaction was only available following local rearrangement of the side chains of the active site because of inhibitor binding. This interaction with Tyr55 was not seen in past hDAAO–ligand cocrystal structures (for example, see Figure 4B). Additional interactions not observed in previous hDAAO–ligand structures included a hydrogen bond between the coumarin hydroxyl and the carbonyl backbone of Gln53 and a water molecule interaction with the cyclic oxygen and carbonyl of compound 2's coumarin ring (Figure 3B).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: *Discovery Research Department, Sunovion Pharmaceuticals, Marlborough, MA 01752, U.S.A.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus