PEG-DAAO conjugate: A promising tool for cancer therapy optimized by protein engineering.

The flavoenzyme D-amino acid oxidase (DAAO) represents a potentially good option for cancer enzyme prodrug therapy as it produces H2O2 using D-amino acids as substrates, compounds present at low concentration in vivo and that can be safely administered to regulate H2O2 production. We optimized the cytotoxicity of the treatment by: i) using an efficient enzyme variant active at low O2 and D-alanine concentrations (mDAAO); ii) improving the stability and half-life of mDAAO and the enhanced permeability and retention effect by PEGylation; and iii) inhibiting the antioxidant cellular system by a heme oxygenase-1 inhibitor (ZnPP). A very low amount of PEG-mDAAO (10 mU, 50 ng of enzyme) induces cytotoxicity on various tumor cell lines. Notably, PEG-mDAAO seems well suited for in vivo evaluation as it shows the same cytotoxicity at air saturation (21%) and 2.5% O2, a condition resembling the microenvironment found in the central part of tumors.

Drug discovery strategies and the preclinical development of D-amino-acid oxidase inhibitors as antipsychotic therapies.

INTRODUCTION: D-amino-acid oxidase (DAAO) degrades D-serine, a co-agonist of the NMDA receptor whose dysfunction is involved in the positive, negative, and cognitive symptoms of schizophrenia. The inhibition of DAAO appears to be a viable strategy to increase D-serine level and to have therapeutic potential in schizophrenia. Areas covered: This review describes the efforts to develop DAAO inhibitors and to optimize their in vitro and in vivo effects in preclinical settings. The structural evolution of DAAO inhibitors is presented from simple carboxylic acid derivatives via small, planar compounds with carboxylic acid mimetics to extended compounds whose binding is possible owing to DAAO flexibility. Inhibitory potency and pharmacokinetic properties are discussed in the context of compounds' ability to increase D-serine level and to show efficacy in animal models of schizophrenia. Expert opinion: The accumulated knowledge on the structural requirements of DAAO inhibitors and on their in vitro and in vivo effects provides appropriate basis to develop inhibitors with optimized potency, selectivity and pharmacokinetic profile including blood-brain penetration. In addition, the validation of DAAO inhibition therapy in alleviating the symptoms of schizophrenia requires further studies on the efficacy of DAAO inhibitors in behavioral assays of animals and on the species differences in D-serine metabolism.

Selenoprotein W as a molecular target of d-amino acid oxidase is regulated by d-amino acid in chicken neurons.

Selenoprotein W (SelW) is an important member of the avian selenoprotein family. It is well known for its important role in protecting neurons from oxidative stress during neuronal development. d-Amino acid (d-serine), as a neurotransmitter in the central nervous system (CNS), can mediate neurotoxicity. d-Amino acid oxidase (DAAO) is responsible for regulating the d-serine levels in cells. However, the correlation between SelW and DAAO is not clear yet. To investigate the regulations between SelW and DAAO, chicken embryo monolayer neurons were treated with d-serine and/or Se. In this study, we predicted molecular binding between SelW and DAAO. These results showed that the 9-16, 18, 41-47 and 66 residues of SelW could combine with the DAAO, which suggested that chicken SelW might be the target of DAAO. We determined the DAAO activity and the mRNA expression of SelW in in vitro cultured chicken embryo primitive neuron cells. d-Serine influenced the activity of DAAO and, moreover, a significant increase in the mRNA expression of SelW was found in neurons treated with Se. Notably, we also observed changes in the expression of SelW and DAAO when neurons were treated with various concentrations of d-serine and Se. In conclusion, these data suggest that d-serine could regulate the mRNA expression of SelW by interfering with the activity of DAAO in chicken embryo neurons.

Discovery of d-amino acid oxidase inhibitors based on virtual screening against the lid-open enzyme conformation.

d-Amino acid oxidase (DAAO) inhibitors are typically small polar compounds with often suboptimal pharmacokinetic properties. Features of the native binding site limit the operational freedom of further medicinal chemistry efforts. We therefore initiated a structure based virtual screening campaign based on the X-ray structures of DAAO complexes where larger ligands shifted the loop (lid opening) covering the native binding site. The virtual screening of our in-house collection followed by the in vitro test of the best ranked compounds led to the identification of a new scaffold with micromolar IC50. Subsequent SAR explorations enabled us to identify submicromolar inhibitors. Docking studies supported by in vitro activity measurements suggest that compounds bind to the active site with a salt-bridge characteristic to DAAO inhibitor binding. In addition, displacement of and interaction with the loop covering the active site contributes significantly to the activity of the most potent compounds.

Intramuscular Delivery of scAAV9-hIGF1 Prolongs Survival in the hSOD1G93A ALS Mouse Model via Upregulation of D-Amino Acid Oxidase.

Self-complementary adeno-associated viral vector 9 (scAAV9) has been confirmed to be an efficient AAV serotype for gene transfer to the central nervous system (CNS). Neurotrophic factors have been considered to be therapeutic targets for amyotrophic lateral sclerosis (ALS). In the present study, we intramuscularly injected scAAV9 encoding human insulin-like growth factor 1 (hIGF1) into an hSOD1G93A ALS mouse model. We observed that scAAV9-hIGF1 significantly reduced the loss of motor neurons of the anterior horn in the lumbar spinal cord and delayed muscle atrophy in ALS mice. Importantly, IGF1 significantly delayed disease onset and prolonged the life span of ALS mice. In addition, scAAV9-hIGF1 protected motor neurons from apoptosis through upregulation of D-amino acid oxidase (DAO), which controls the level of D-serine. Moreover, to further verify these results, we used CRISPR-Cas9 system to target the central nervous system knockdown of IGF1. This experiment supported the continued investigation of neurotrophic factor gene therapies targeting the central nervous system as a potential treatment for ALS.

Selection and characterization of alanine racemase inhibitors against Aeromonas hydrophila.

BACKGROUND: Combining experimental and computational screening methods has been of keen interest in drug discovery. In the present study, we developed an efficient screening method that has been used to screen 2100 small-molecule compounds for alanine racemase Alr-2 inhibitors. RESULTS: We identified ten novel non-substrate Alr-2 inhibitors, of which patulin, homogentisic acid, and hydroquinone were active against Aeromonas hydrophila. The compounds were found to be capable of inhibiting Alr-2 to different extents with 50% inhibitory concentrations (IC50) ranging from 6.6 to 17.7 µM. These compounds inhibited the growth of A. hydrophila with minimal inhibitory concentrations (MICs) ranging from 20 to 120 µg/ml. These compounds have no activity on horseradish peroxidase and D-amino acid oxidase at a concentration of 50 µM. The MTT assay revealed that homogentisic acid and hydroquinone have minimal cytotoxicity against mammalian cells. The kinetic studies indicated a competitive inhibition of homogentisic acid against Alr-2 with an inhibition constant (K i) of 51.7 µM, while hydroquinone was a noncompetitive inhibitor with a K i of 212 µM. Molecular docking studies suggested that homogentisic acid binds to the active site of racemase, while hydroquinone lies near the active center of alanine racemase. CONCLUSIONS: Our findings suggested that combining experimental and computational methods could be used for an efficient, large-scale screening of alanine racemase inhibitors against A. hydrophila that could be applied in the development of new antibiotics against A. hydrophila.

Fabrication of enzyme reactor utilizing magnetic porous polymer membrane for screening D-Amino acid oxidase inhibitors.

In this work, a unique D-amino acid oxidase reactor for enhanced enzymolysis efficiency is presented. A kind of magnetic polymer matrices, composed of iron oxide nanoparticles and porous polymer membrane (poly styrene-co-maleic anhydride), was prepared. With covalent bonding D-Amino acid oxidase on the surface of the matrices and characterization of scanning electron microscope and vibrating sample magnetometer, it demonstrated that the membrane enzyme reactor was successfully constructed. The enzymolysis efficiency of the enzyme reactor was evaluated and the apparent Michaelis-Menten constants of D-Amino acid oxidase were determined (Km was 1.10mM, Vmax was 23.8mMmin-1) by a chiral ligand exchange capillary electrophoresis protocol with methionine as the substrate. The results indicated that the enzyme reactor could exhibit good stability and excellent reusability. Importantly, because the enzyme and the substrate could be confined into the pores of the matrices, the enzyme reactor displayed the improved enzymolysis efficiency due to the confinement effect. Further, the prepared enzyme reactor was applied for D-Amino acid oxidase inhibitors screening. It has displayed that the proposed protocol could pave a new way for fabrication of novel porous polymer membrane based enzyme reactors to screen enzyme inhibitors.

Construction of a D-amino acid oxidase reactor based on magnetic nanoparticles modified by a reactive polymer and its application in screening enzyme inhibitors.

Developing facile and high-throughput methods for exploring pharmacological inhibitors of D-amino acid oxidase (DAAO) has triggered increasing interest. In this work, DAAO was immobilized on the magnetic nanoparticles, which were modified by a biocompatible reactive polymer, poly(glycidyl methacrylate) (PGMA) via an atom transfer radical polymerization technique. Interestingly, the enzyme immobilization process was greatly promoted with the assistance of a lithium perchlorate catalyst. Meanwhile, a new amino acid ionic liquid (AAIL) was successfully synthesized and employed as the efficient chiral ligand in a chiral ligand exchange capillary electrophoresis (CLE-CE) system for chiral separation of amino acids (AAs) and quantitation of methionine, which was selected as the substrate of DAAO. Then, the apparent Michaelis-Menten constants in the enzyme system were determined with the proposed CLE-CE method. The prepared DAAO-PGMA-Fe3O4 nanoparticles exhibited excellent reusability and good stability. Moreover, the enzyme reactor was successfully applied in screening DAAO inhibitors. These results demonstrated that the enzyme could be efficiently immobilized on the polymer-grafted magnetic nanoparticles and that the obtained enzyme reactor has great potential in screening enzyme inhibitors, further offering new insight into monitoring the relevant diseases.

4-Hydroxypyridazin-3(2H)-one derivatives as novel D-amino acid oxidase inhibitors.

D-Amino acid oxidase (DAAO) catalyzes the oxidation of d-amino acids including d-serine, a coagonist of the N-methyl-d-aspartate receptor. We identified a series of 4-hydroxypyridazin-3(2H)-one derivatives as novel DAAO inhibitors with high potency and substantial cell permeability using fragment-based drug design. Comparisons of complex structures deposited in the Protein Data Bank as well as those determined with in-house fragment hits revealed that a hydrophobic subpocket was formed perpendicular to the flavin ring by flipping Tyr224 in a ligand-dependent manner. We investigated the ability of the initial fragment hit, 3-hydroxy-pyridine-2(1H)-one, to fill this subpocket with the aid of complex structure information. 3-Hydroxy-5-(2-phenylethyl)pyridine-2(1H)-one exhibited the predicted binding mode and demonstrated high inhibitory activity for human DAAO in enzyme- and cell-based assays. We further designed and synthesized 4-hydroxypyridazin-3(2H)-one derivatives, which are equivalent to the 3-hydroxy-pyridine-2(1H)-one series but lack cell toxicity. 6-[2-(3,5-Difluorophenyl)ethyl]-4-hydroxypyridazin-3(2H)-one was found to be effective against MK-801-induced cognitive deficit in the Y-maze.

Pharmacodynamic effects of a D-amino acid oxidase inhibitor indicate a spinal site of action in rat models of neuropathic pain.

Inhibition of d-amino acid oxidase (DAAO) activity is a potential target for the treatment of chronic pain. Here we characterized the effects of systemic administration of the DAAO inhibitor 4H-furo[3,2-b]pyrrole-5-carboxylic acid (SUN) in rat models of neuropathic and inflammatory pain. Oral administration of SUN dose dependently attenuated tactile allodynia induced by ligation of the L5 spinal nerve (SNL) and similarly reversed thermal hyperalgesia produced by chronic constriction injury. In addition, SUN was efficacious against complete Freund's adjuvant-induced thermal hyperalgesia. In these models, maximal reversal of pain-related behaviors corresponded with maximum rates of increase in brain and plasma d-serine concentrations, indicative of full inhibition of DAAO activity. To investigate the possible site(s) of action, we recorded spontaneous nerve activity and mechanically evoked responses of central spinal cord dorsal horn neurons and compared these with spontaneous activity of peripheral dorsal root filaments in anesthetized SNL model animals. Oral SUN reduced spontaneous activity in both central and peripheral recordings at doses and pretreatment times that corresponded to reduced mechanical allodynia in behavioral experiments. After intravenous administration of SUN, the onset of action for this central effect was rapid (maximal effects within 30 minutes), but was abolished by severing afferent inputs to the dorsal horn. Overall, these results indicate that inhibition of DAAO in peripheral afferent spinal circuits reduced spontaneous neuronal activity to attenuate pain-related behaviors in rat models of neuropathic and inflammatory pain.

Identification of novel D-amino acid oxidase inhibitors by in silico screening and their functional characterization in vitro.

D-amino acid oxidase (DAO) is a degradative enzyme that is stereospecific for D-amino acids, including D-serine and D-alanine, which are potential coagonists of the N-methyl-D-aspartate (NMDA) receptor. Dysfunction of NMDA receptor-mediated neurotransmission has been implicated in the onset of various mental disorders such as schizophrenia. Hence, a DAO inhibitor that augments the brain levels of D-serine and/or D-alanine and thereby activates NMDA receptor function is expected to be an antipsychotic drug, for instance, in the treatment of schizophrenia. In the search for potent DAO inhibitor(s), a large number of compounds were screened in silico, and several compounds were estimated as candidates. These compounds were then characterized and evaluated as novel DAO inhibitors in vitro. The results reported in this study indicate that some of these compounds are possible lead compounds for the development of a clinically useful DAO inhibitor and have the potential to serve as active site probes to elucidate the structure-function relationships of DAO.

A novel chiral ligand exchange capillary electrophoresis system with amino acid ionic liquid as ligand and its application in screening D-amino-acid oxidase inhibitors.

A novel amino acid ionic liquid (AAIL) with L-ornithine (L-Orn) as anion was successfully synthesized, and subsequently applied as an available chiral ligand coordinated with Zn(II) in a chiral ligand exchange capillary electrophoresis (CLE-CE) system for the enantioseparation of dansyl amino acids (Dns-D,L-AAs). The influence of key parameters, such as buffer pH, concentration ratio of Zn(II) to ligand and complex concentration, was investigated in detail. Eleven pairs of Dns-D,L-AAs enantiomers were baseline separated and three pairs were partly separated under the optimum conditions. For exploring its potential application, the quantitative features of this proposed method were studied. Good linearity (r(2) = 0.999) and favorable repeatability (RSD ≤ 3.4%) were obtained by using Dns-D,L-Met as the test analyte. Finally, this method was employed to investigate the inhibition efficiency of d-amino acid oxidase (DAAO) inhibitors, which may pave a new way for the high-throughput screening of enzyme inhibitors and relevant drug discovery.

Modulation of NMDA receptor function by inhibition of D-amino acid oxidase in rodent brain.

Observations that N-Methyl-D-Aspartate (NMDA) antagonists produce symptoms in humans that are similar to those seen in schizophrenia have led to the current hypothesis that schizophrenia might result from NMDA receptor hypofunction. Inhibition of D-amino acid oxidase (DAAO), the enzyme responsible for degradation of D-serine, should lead to increased levels of this co-agonist at the NMDA receptor, and thereby provide a therapeutic approach to schizophrenia. We have profiled some of the preclinical biochemical, electrophysiological, and behavioral consequences of administering potent and selective inhibitors of DAAO to rodents to begin to test this hypothesis. Inhibition of DAAO activity resulted in a significant dose and time dependent increase in D-serine only in the cerebellum, although a time delay was observed between peak plasma or brain drug concentration and cerebellum D-serine response. Pharmacokinetic/pharmacodynamic (PK/PD) modeling employing a mechanism-based indirect response model was used to characterize the correlation between free brain drug concentration and D-serine accumulation. DAAO inhibitors had little or no activity in rodent models considered predictive for antipsychotic activity. The inhibitors did, however, affect cortical activity in the Mescaline-Induced Scratching model, produced a modest but significant increase in NMDA receptor-mediated synaptic currents in primary neuronal cultures from rat hippocampus, and resulted in a significant increase in evoked hippocampal theta rhythm, an in vivo electrophysiological model of hippocampal activity. These findings demonstrate that although DAAO inhibition did not cause a measurable increase in D-serine in forebrain, it did affect hippocampal and cortical activity, possibly through augmentation of NMDA receptor-mediated currents.

Inhibition of D-amino acid oxidase activity by antipsychotic drugs evaluated by a fluorometric assay using D-kynurenine as substrate.

A facile fluorometric assay using D-kynurenine as a substrate was utilized for evaluating the inhibition of D-amino acid oxidase (DAAO), which is one of the products of a susceptibility gene for schizophrenia, by commercial antipsychotic drugs, namely, chlorpromazine (CPZ), carbamazepine, sulpiride, quetiapine, and imipramine. CPZ inhibited DAAO (65.8 ± 13.2 µM, n = 3) as reported previously, and other drugs also inhibited DAAO activity. Among these, quetiapine had the smallest IC(50) value (19.5 ± 2.60 µM, n = 3). The proposed assay can be useful for the evaluation or screening of DAAO-inhibitory drugs.

D-glufosinate as a male sterility agent for hybrid seed production.

A chemical male sterility system based on anther-localized conversion of the inactive D-enantiomer of the herbicide, glufosinate (2-amino-4-(methylphosphinyl)-butanoate) to the phytotoxic L is described. Highly pure D-glufosinate was isolated in >98% enantiomeric excess from the racemate via fermentation with a strain of Escherichia coli expressing the PAT (L-glufosinate N-acetyl transferase) gene and purification of the unreacted D-enantiomer from the broth by ion exchange. A modified (F58K, M213S) form of the D-amino acid oxidase (DAAO) (EC 1.4.3.3) from Rhodosporidium toruloides was designed, tested in vitro and found to efficiently oxidize D-glufosinate to its 2-oxo derivative [2-oxo-4-(methylphosphinyl)-butanoic acid]. Tobacco (Nicotiana tabacum) plants were transformed to express this modified oxidase under control of the TAP1 tapetum-specific promoter. A number of the resultant transgenic lines exhibited complete male sterility that persisted for two or more weeks immediately following foliar treatment with 75 or 200 g/ha of D-glufosinate without exhibiting obvious phytotoxic symptoms or any measurable decline in female fertility. Similarly, plants containing the same construct and, additionally, a PAT gene expressed from a plastocyanin promoter exhibited significantly reduced male fertility and no reduction in female fertility following foliar application of racemic glufosinate. Thus, foliar application of d-glufosinate either purified or as the commercial herbicide, combined with anther expression of a modified DAAO promises to provide a cost-effective conditional chemical male sterility system with the characteristics necessary for practical F1 hybrid seed production.

Increased D-amino acid oxidase expression in the bilateral hippocampal CA4 of schizophrenic patients: a post-mortem study.

An important risk gene in schizophrenia is D-: amino acid oxidase (DAAO). To establish if expression of DAAO is altered in cortical, hippocampal or thalamic regions of schizophrenia patients, we measured gene expression of DAAO in a post-mortem study of elderly patients with schizophrenia and non-affected controls in both hemispheres differentiating between gray and white matter. We compared cerebral post-mortem samples (granular frontal cortex BA9, middle frontal cortex BA46, superior temporal cortex BA22, entorhinal cortex BA28, sensoric cortex BA1-3, hippocampus (CA4), mediodorsal nucleus of the thalamus) from 10 schizophrenia patients to 13 normal subjects investigating gene expression of DAAO in the gray and white matter of both hemispheres of the above-mentioned brain regions by in situ-hybridization. We found increased expression of DAAO-mRNA in the hippocampal CA4 of schizophrenic patients. Compared to the control group, both hemispheres of the hippocampus of schizophrenic patients showed an increased expression of 46% (right, P = 0.013) and 54% (left, P = 0.019), respectively. None of the other regions examined showed statistically significant differences in DAAO expression. This post-mortem study demonstrated increased gene expression of DAAO in the left and right hippocampus of schizophrenia patients. This increased expression could be responsible for a decrease in local D-: serine levels leading to a NMDA-receptor hypofunction that is hypothesized to play a major role in the pathophysiology of schizophrenia. However, our study group was small and results should be verified using larger samples.

Optimization of D-amino acid oxidase for low substrate concentrations--towards a cancer enzyme therapy.

D-amino acid oxidase (DAAO) has recently become of interest as a biocatalyst for industrial applications and for therapeutic treatments. It has been used in gene-directed enzyme prodrug therapies, in which its production of H2O2 in tumor cells can be regulated by administration of substrate. This approach is limited by the locally low O2 concentration and the high K(m) for this substrate. Using the directed evolution approach, one DAAO mutant was identified that has increased activity at low O2 and D-Ala concentrations and a 10-fold lower K(m) for O2. We report on the mechanism of this DAAO variant and on its cytotoxicity towards various mammalian cancer cell lines. The higher activity observed at low O2 and D-Ala concentrations results from a combination of modifications of specific kinetic steps, each being of small magnitude. These results highlight the potential in vivo applicability of this evolved mutant DAAO for tumor therapy.

Discovery, SAR, and pharmacokinetics of a novel 3-hydroxyquinolin-2(1H)-one series of potent D-amino acid oxidase (DAAO) inhibitors.

3-Hydroxyquinolin-2(1H)-one (2) was discovered by high throughput screening in a functional assay to be a potent inhibitor of human DAAO, and its binding affinity was confirmed in a Biacore assay. Cocrystallization of 2 with the human DAAO enzyme defined the binding site and guided the design of new analogues. The SAR, pharmacokinetics, brain exposure, and effects on cerebellum D-serine are described. Subsequent evaluation against the rat DAAO enzyme revealed a divergent SAR versus the human enzyme and may explain the high exposures of drug necessary to achieve significant changes in rat or mouse cerebellum D-serine.

Molecular cloning and expression in Escherichia coli of an active fused Zea mays L. D-amino acid oxidase.

D-Amino acid oxidase (DAAO) is an FAD-dependent enzyme that metabolizes D-amino acids in microbes and animals. However, such ability has not been identified in plants so far. We predicted a complete DAAO coding sequence consisting of 1158 bp and encoding a protein of 386 amino acids. We cloned this sequence from the leaf cDNA population of maize plants that could utilize D-alanine as a nitrogen source and grow normally on media containing D-Ala at the concentrations of 100 and 1000 ppm. For more understanding of DAAO ability in maize plant, we produced a recombinant plasmid by the insertion of isolated cDNA into the pMALc2X Escherichia coli expression vector, downstream of the maltose-binding protein coding sequence. The pMALc2X-DAAO vector was used to transform the TB1 strain of E. coli cells. Under normal growth conditions, fused DAAO (with molecular weight of about 78 kDa) was expressed up to 5 mg/liter of bacterial cells. The expressed product was purified by affinity chromatography and subjected to in vitro DAAO activity assay in the presence of five different D-amino acids. Fused DAAO could oxidize D-alanine and D-aspartate, but not D-leucine, D-isoleucine, and D-serine. The cDNA sequence reported in this paper has been submitted to EMBL databases under accession number AM407717.

Inhibition of D-amino-Acid oxidase activity induces pain relief in mice.

(1). We investigated the effects of inhibiting D: -amino-acid oxidase (DAO) activity on nociceptive responses through the use of mutant ddY/DAO(-) mice, which lack DAO activity, and through the application of a selective inhibitor of DAO, sodium benzoate, in the tail flick test, hot-plate test, formalin test, and acetic acid-induced writhing test. (2). Compared with normal ddY/DAO+ mice, ddY/DAO(- )mice showed significantly prolonged tail withdrawal latency in the tail flick test and licking/jumping latency in the hot-plate test, as well as significantly reduced duration of licking/biting in the late phase of the formalin test and the number of abdominal writhing in the acetic acid-induced writhing test. (3). In addition, we investigated the effects of sodium benzoate in Kunming mice having normal DAO activity. (4). Intravenous administration of sodium benzoate (400 mg/kg) significantly inhibited pain responses of the late phase of the formalin test and abdominal writhing responses in the acetic acid-induced writhing test, with no effects on the early phase flinch responses in the formalin test, nociceptive responses in the tail flick test, or hot-plate test. (5). These results suggest that DAO acts as a pro-nociceptive factor in pain, particularly chronic pain, transmission and modulation, and may be a target for pain treatment.