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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.

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Profiling compounds in enzymatic inhibitory assays(A) In the Amplex Red platform, compound (cpd) 1 and cpd 2 were tested for hDAAO inhibition with D-serine as substrate (solid lines) and counter assay inhibition (dashed lines). Neither compound produced any inhibition in the counter assay. (B) Cpd 1 and cpd 2 inhibited hDAAO in the hDAAO inhibition assay with D-phenylglycine as substrate and using LC–MS for direct product detection. (C) Cpd 1 and cpd 2 inhibited rDAAO, albeit less potently than they inhibited hDAAO. (D) Jump-dilution assay example data for cpd 2. In the Amplex Red platform, activity of hDAAO was monitored kinetically as production of fluorescent product over time. In the ‘koff’ condition (squares; ‘200 nM then 5 nM’), 200 nM cpd 2 (an inhibitory concentration; see Figure 2A) was rapidly diluted to 5 nM (a non-inhibitory concentration). Initially hDAAO was inhibited as reported by a shallow slope of product production defined as low initial velocity (vi). Over time, inhibitor dissociates and a more rapid, steady-state velocity of product production (vs) was observed. Quantitative data from each of these assays are presented in Table 1.
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Figure 1: Profiling compounds in enzymatic inhibitory assays(A) In the Amplex Red platform, compound (cpd) 1 and cpd 2 were tested for hDAAO inhibition with D-serine as substrate (solid lines) and counter assay inhibition (dashed lines). Neither compound produced any inhibition in the counter assay. (B) Cpd 1 and cpd 2 inhibited hDAAO in the hDAAO inhibition assay with D-phenylglycine as substrate and using LC–MS for direct product detection. (C) Cpd 1 and cpd 2 inhibited rDAAO, albeit less potently than they inhibited hDAAO. (D) Jump-dilution assay example data for cpd 2. In the Amplex Red platform, activity of hDAAO was monitored kinetically as production of fluorescent product over time. In the ‘koff’ condition (squares; ‘200 nM then 5 nM’), 200 nM cpd 2 (an inhibitory concentration; see Figure 2A) was rapidly diluted to 5 nM (a non-inhibitory concentration). Initially hDAAO was inhibited as reported by a shallow slope of product production defined as low initial velocity (vi). Over time, inhibitor dissociates and a more rapid, steady-state velocity of product production (vs) was observed. Quantitative data from each of these assays are presented in Table 1.

Mentions: We utilized a novel screening strategy to identify compound 2, a potent inhibitor of hDAAO (See Supplementary Online Data; compound 2 structure in Table 1). To confirm that compound 2 was a reversible inhibitor of hDAAO, we profiled it in a series of biochemical assays, using the previously characterized hDAAO inhibitor compound 1 [22,27] as positive control. As shown in Figure 1(A), compounds 1 and 2 inhibited hDAAO activity. However, they did not display inhibition in the counter assay, verifying that these compounds did not interfere with the assay detection system non-specifically. To fully exclude fluorescence artefacts that could arise in the Amplex Red system, we developed an orthogonal assay in which the direct product of the hDAAO oxidative reaction was measured by LC–MS, when D-phenylglycine was used as substrate. In this assay, both compounds 1 and 2 were confirmed as potent hDAAO inhibitors (Figure 1B and Table 1). Both compounds inhibited rDAAO, although the compounds were less potent as inhibitors of rDAAO compared with hDAAO (Figure 1C and Table 1). Compound 1 and other hDAAO inhibitors have previously been observed to be less potent rDAAO inhibitors [22]. To confirm that the compounds were reversible, we utilized a jump-dilution assay protocol, in which the inhibited enzyme–ligand complexes were diluted to allow for kinetic measurement of inhibitor dissociation and reconstitution of enzyme activity [38]. For compound 2, we pre-incubated in 200 nM (a concentration yielding >80% inhibition; see Figure 1A), and then after dilution the compound was at 5 nM (no significant inhibition under 50 mM D-serine recovery conditions). Reaction product was recorded immediately after dilution (Figure 1D). Under these conditions, initial velocity (Vi) was low, but over time, inhibitor dissociated and a steady-state velocity (Vs) was reached. These results were consistent with reversible inhibition [38]. The recovery time course was fit with equation (1) (the Experimental section) to obtain an apparent rate constant for dissociation (koff) as reported in Table 1.


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)

Profiling compounds in enzymatic inhibitory assays(A) In the Amplex Red platform, compound (cpd) 1 and cpd 2 were tested for hDAAO inhibition with D-serine as substrate (solid lines) and counter assay inhibition (dashed lines). Neither compound produced any inhibition in the counter assay. (B) Cpd 1 and cpd 2 inhibited hDAAO in the hDAAO inhibition assay with D-phenylglycine as substrate and using LC–MS for direct product detection. (C) Cpd 1 and cpd 2 inhibited rDAAO, albeit less potently than they inhibited hDAAO. (D) Jump-dilution assay example data for cpd 2. In the Amplex Red platform, activity of hDAAO was monitored kinetically as production of fluorescent product over time. In the ‘koff’ condition (squares; ‘200 nM then 5 nM’), 200 nM cpd 2 (an inhibitory concentration; see Figure 2A) was rapidly diluted to 5 nM (a non-inhibitory concentration). Initially hDAAO was inhibited as reported by a shallow slope of product production defined as low initial velocity (vi). Over time, inhibitor dissociates and a more rapid, steady-state velocity of product production (vs) was observed. Quantitative data from each of these assays are presented in Table 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Profiling compounds in enzymatic inhibitory assays(A) In the Amplex Red platform, compound (cpd) 1 and cpd 2 were tested for hDAAO inhibition with D-serine as substrate (solid lines) and counter assay inhibition (dashed lines). Neither compound produced any inhibition in the counter assay. (B) Cpd 1 and cpd 2 inhibited hDAAO in the hDAAO inhibition assay with D-phenylglycine as substrate and using LC–MS for direct product detection. (C) Cpd 1 and cpd 2 inhibited rDAAO, albeit less potently than they inhibited hDAAO. (D) Jump-dilution assay example data for cpd 2. In the Amplex Red platform, activity of hDAAO was monitored kinetically as production of fluorescent product over time. In the ‘koff’ condition (squares; ‘200 nM then 5 nM’), 200 nM cpd 2 (an inhibitory concentration; see Figure 2A) was rapidly diluted to 5 nM (a non-inhibitory concentration). Initially hDAAO was inhibited as reported by a shallow slope of product production defined as low initial velocity (vi). Over time, inhibitor dissociates and a more rapid, steady-state velocity of product production (vs) was observed. Quantitative data from each of these assays are presented in Table 1.
Mentions: We utilized a novel screening strategy to identify compound 2, a potent inhibitor of hDAAO (See Supplementary Online Data; compound 2 structure in Table 1). To confirm that compound 2 was a reversible inhibitor of hDAAO, we profiled it in a series of biochemical assays, using the previously characterized hDAAO inhibitor compound 1 [22,27] as positive control. As shown in Figure 1(A), compounds 1 and 2 inhibited hDAAO activity. However, they did not display inhibition in the counter assay, verifying that these compounds did not interfere with the assay detection system non-specifically. To fully exclude fluorescence artefacts that could arise in the Amplex Red system, we developed an orthogonal assay in which the direct product of the hDAAO oxidative reaction was measured by LC–MS, when D-phenylglycine was used as substrate. In this assay, both compounds 1 and 2 were confirmed as potent hDAAO inhibitors (Figure 1B and Table 1). Both compounds inhibited rDAAO, although the compounds were less potent as inhibitors of rDAAO compared with hDAAO (Figure 1C and Table 1). Compound 1 and other hDAAO inhibitors have previously been observed to be less potent rDAAO inhibitors [22]. To confirm that the compounds were reversible, we utilized a jump-dilution assay protocol, in which the inhibited enzyme–ligand complexes were diluted to allow for kinetic measurement of inhibitor dissociation and reconstitution of enzyme activity [38]. For compound 2, we pre-incubated in 200 nM (a concentration yielding >80% inhibition; see Figure 1A), and then after dilution the compound was at 5 nM (no significant inhibition under 50 mM D-serine recovery conditions). Reaction product was recorded immediately after dilution (Figure 1D). Under these conditions, initial velocity (Vi) was low, but over time, inhibitor dissociated and a steady-state velocity (Vs) was reached. These results were consistent with reversible inhibition [38]. The recovery time course was fit with equation (1) (the Experimental section) to obtain an apparent rate constant for dissociation (koff) as reported in Table 1.

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