D-amino Acid Oxidase Research Articles

Novel Enzymatic Method for Imine Synthesis via the Oxidation of Primary Amines Using D-Amino Acid Oxidase from Porcine Kidney

During studies on the oxidative cyanation reaction catalyzed by a variant of D-amino acid oxidase from porcine kidney (pkDAO) (Y228L/R283G), an unexpected formation of 1-phenyl-N-(1-phenylethylidene)ethanamine (PPEA) was detected. The optimal reaction conditions for the synthesis of PPEA and the reaction mechanism were investigated using the pkDAO variant. The highest PPEA synthesis was obtained in the reaction with 150 mM (R)-MBA at pH 9.0 and at 20 °C. Since PPEA synthesis proceeded by trapping the intermediate 1-phenylethanimine (1-PEI) by 15N-labeled n-hexylamine, which is not a substrate for the pKDAO variant, it was deduced that PPEA would be synthesized by a nucleophilic substitution of 1-PEI by another molecule of (R)-MBA. PPEA was further identified by its conversion to bis(1-phenylethyl)amine (BPEA) through reduction with NaBH4. Thus, a new enzymatic method of imine synthesis by oxidation of primary amine by the variant pkDAO was achieved for the first time.

MO040: Development of BBP-711, a small molecule inhibitor of glycolate oxidase for primary hyperoxaluria type 1 and recurrent kidney stone formers

Abstract BACKGROUND AND AIMS BBP-711 is an investigational small molecule inhibitor of glycolate oxidase (GO), currently in development for the treatment of primary hyperoxaluria type 1 (PH1) and recurrent nephrolithiasis. Selection of BBP-711 as a drug candidate followed structural activity optimization and a pharmacological testing strategy that included functional activity in enzyme assays, target engagement in cellular assays and efficacy studies in a rodent model (Agxt−/− mice) of primary hyperoxaluria type 1. METHOD BBP-711 was developed by iterative synthesis and study of the structure–activity relationship of more than 50 GO inhibitors. Lead molecules including BBP-711 were assessed for functional activity by determining inhibition of the conversion of glycolate to oxalate with purified human, mouse and rat HAOX1 enzymes. The ability of BBP-711 to bind directly to immobilized purified hGO (HAOX1 gene product) was evaluated by surface plasmon resonance. Selectivity of BBP-711 was assessed by determining its inhibition of several physiologically related gene products including that from HAOX2. In vitro efficacy of BBP-711 was tested via inhibition of oxalate production in primary hepatocytes cultured from hyperoxaluric Agxt−/− mice. In vivo efficacy of BBP-711 on urinary oxalate excretion was assessed at various doses given by oral gavage in Agxt −/− mice. Pharmacokinetics of BBP-711 was investigated to assess plasma and liver concentrations associated with efficacy. RESULTS BBP-711 is a potent inhibitor of human, rat and mouse GO (IC50 = 15.4 nM, 22.4 nM and 149 nM, respectively). BBP-711 demonstrated stronger direct binding to hGO1 (KD = 6.31 nM) than to rGO2 (KD = 12.8 nM), indicating its preference for GO1 as a target. BBP-711 specificity was demonstrated with <10% activity against off-target enzymes D-amino acid oxidase, dihydroorotate dehydrogenase and lactate dehydrogenase-A at 10 µM. BBP-711 demonstrated concentration-dependent inhibition of oxalate production by Agxt−/− hepatocytes (IC50 = 24.2 nM at 24 hours, IC50 = 42.9 nM at 48 hours). When orally dosed at 7 mg/kg for 5 days in Agxt−/− mice, BBP-711 produced a maximum reduction of urinary oxalate (60%) as well as maximum inhibition of GO activity (>88%). Higher doses did not result in greater reduction in urinary oxalate. Liver exposures (AUC0-24hr) were higher than plasma exposures at all doses tested. GO engagement by BBP-711 was also assessed in an ex vivo assay. Liver extracts from Agxt−/− and wild-type mice dosed with BBP-711 show a dose-dependent reduction in ex vivo GO activity at 1 and 4-hour post-dose. CONCLUSION Iterative and rational optimization of small molecule GO inhibitors resulted in BBP-711, a potent and specific inhibitor of GO with drug-like properties.

Hydrogen sulfide has a role as an endogenous relaxation factor in the rat prostate

ABSTRACT Introduction Hydrogen sulfide (H2S) is the third endogenous gasotransmitter besides carbon monoxide and nitric oxide, and has a wide range of physiological functions such as neuromodulation, vasorelaxation and cytoprotection. Although in human prostate cancer cells, NaHS (a H2S donor) treatment was reported to inhibit proliferation and to elevate secretory function, physiological functions of H2S in the prostate tissue had not been investigated yet. Endogenous H2S is biosynthesized from L-cysteine by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). In addition, 3-mercaptopyruvate sulfurtransferase (MPST) synthesizes H2S from 3-mercaptopyruvate (3MP), which is produced from L-cysteine by cysteine aminotransferase (CAT) (CAT/MPST pathway). Recently, a new pathway for endogenous H2S biosynthesis is reported; D-cysteine is metabolized by D-amino acid oxidase (DAO) to 3MP, which is a substrate for MPST to synthesize H2S. Objective We investigated (1) tissue distribution of CBS, CSE, MPST, CAT and DAO, (2) endogenous H2S levels, and (3) effects of NaHS on contractility in the rat prostate. Methods Dorsolateral and ventral prostate (PR-D and PR-V), liver and cerebellum (L and C) were prepared from male Wistar rats (300-400 g) sacrificed with an overdose of sodium pentobarbital (80 mg/kg, ip). (1) Tissue distributions of 5 enzymes were investigated by real-time PCR, western blot and immunohistochemistry. L and C were used for positive controls. (2) H2S contents in the prostate tissues were measured by methylene blue method. (3) By using 1 x 5 mm strips of the prostate tissues, effects of NaHS (1 x 10-9 to 3 x 10-4 M) were evaluated on pre-contracted prostate strips by noradrenaline (10-5 M). Prostate strips were pretreated with propranolol (10-6 M) 30 min before the pre-contraction. Results (1) CBS, MPST and CAT were detected in the prostate. Expression levels of CBS in the PR-D were lower than those in the PR-V, and the levels of MPST in the PR-D were higher than those in the PR-V. In expression levels of CAT, there was no significant difference between two prostate tissues. On the other hand, CSE or DAO was not detected in these tissues. Immunoreactvities of these enzymes were mainly detected in the prostate glandular epithelium. (2) H2S was detected in the prostate tissues. (3) NaHS dose-dependently induced relaxation of pre-contracted PR-D and PR-V strips. There were no significant differences of the EC50 values or relaxation rate against the pre-contractions between two prostate tissues. Conclusion H2S synthesized by CBS- and CAT/MPST-mediated pathways has a role as an endogenous relaxation factor in the rat prostate. Therefore, endogenous H2S might open new avenues of therapeutic interventions for benign prostatic hyperplasia. Disclosure Work supported by industry: no.

Hydrogen Peroxide Produced in Subcellular Compartments of Lung Adenocarcinoma Cell Line Induces Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is a process by which cancer cells increase invasiveness and motility by changing their phenotype from epithelial to mesenchymal. The mechanism of hydrogen peroxide (H2O2)‐induced EMT remains elusive. We hypothesize that H2O2 production in different subcellular compartments will trigger EMT at different intensities. We used a chemo‐genetic approach to produce hydrogen peroxide in the cytoplasm, nucleus and mitochondria of A549 lung adenocarcinoma cells to see how EMT progresses in each case. Cells were transfected with one of three different plasmids containing specific localization sequences for cytoplasm, nucleus and mitochondria. These plasmids have the peroxide generating enzyme D‐Amino‐Acid Oxidase (DAAO) which generates H2O2 by metabolizing D‐Alanine. We also used the inhibitors of peroxide metabolism, 3‐aminotriazole (3‐AT) and auranofin (Au) to increase H2O2 concentrations in these compartments. Preliminary studies show that cytoplasmic peroxide generation induces EMT significantly after 7 days. The epithelial marker E‐Cadherin was reduced and the mesenchymal marker N‐Cadherin was increased significantly in our immunoassays. The extent of change in EMT markers was greater upon the addition of 3‐AT and Au. However, there was no significant change in EMT markers when H2O2 was generated in the nucleus or mitochondria even in the presence of peroxide metabolism inhibitors. We are investigating the molecular mechanisms responsible for cytoplasmic peroxide‐induced EMT.

Vitreoscilla hemoglobin enhances the catalytic performance of industrial oxidases in vitro.

Oxidases are a group of oxidoreductases and need molecular oxygen in the catalytic process. Vitreoscilla hemoglobin (VHb) can improve the growth and productivity of host cells under hypoxic conditions, rendering it attractive for industrial application. In this work, we demonstrated the addition of immobilized VHb increased the catalytic activity of immobilized D-amino acid oxidase of Trigonopsis variabilis by two-fold when catalyzing cephalosporin C under oxygen-limited conditions. A similar increase of activities was observed in glucose oxidase, alcohol oxidase, and p-hydroxymandelate synthase by adding free VHb or immobilized VHb under hypoxic conditions. When L-glutamate oxidase was used to catalyze L-glutamate to produce α-ketoglutarate, the yield increased from 80.6 to 96.9% by fusing VHb with L-glutamate oxidase. Results demonstrated that the addition of free VHb, immobilized VHb, or fused VHb could increase the catalytic efficiency of oxidases, which was considered by increasing the concentration of the microenvironmental oxygen. Thus, VHb may become a potential additive agent to promote the efficiency of oxidases on industrial scale . KEY POINTS: • First time confirmation of facilitation of VHb on several industrial oxidases in vitro • VHb functions under hypoxic conditions rather than oxygen-enriched conditions • VHb functions in vitro in the form of free, immobilized protein and fusion enzyme.

Discovery of a Novel Class of d-Amino Acid Oxidase Inhibitors Using the Schrödinger Computational Platform.

d-Serine is a coagonist of the N-methyl d-aspartate (NMDA) receptor, a key excitatory neurotransmitter receptor. In the brain, d-serine is synthesized from its l-isomer by serine racemase and is metabolized by the D-amino acid oxidase (DAO, DAAO). Many studies have linked decreased d-serine concentration and/or increased DAO expression and enzyme activity to NMDA dysfunction and schizophrenia. Thus, it is feasible to employ DAO inhibitors for the treatment of schizophrenia and other indications. Powered by the Schrödinger computational modeling platform, we initiated a research program to identify novel DAO inhibitors with the best-in-class properties. The program execution leveraged an hDAO FEP+ model to prospectively predict compound potency. A new class of DAO inhibitors with desirable properties has been discovered from this endeavor. Our modeling technology on this program has not only enhanced the efficiency of structure-activity relationship development but also helped to identify a previously unexplored subpocket for further optimization.

Unveiling the Bio-corona Fingerprinting of Potential Anticancer Carbon Nanotubes Coupled with d-Amino Acid Oxidase

The oxidation therapy, based on the controlled production of Reactive Oxygen Species directly into the tumor site, was introduced as alternative antitumor approach. For this purpose, d-amino acid oxidase (DAAO) from the yeast Rhodotorula gracilis, an enzyme able to efficiently catalyze the production of hydrogen peroxide from d-amino acids, was adsorbed onto multi-walled carbon nanotubes (MWCNTs), previously functionalized with polylactic-co-glycolic acid (PLGA) or polyethylene glycol (PEG) at different degrees to reduce their toxicity, to be targeted directly into the tumor. In vitro activity and cytotoxicity assays demonstrated that DAAO-functionalized nanotubes (f-MWCNTs) produced H2O2 and induced toxic effects to selected tumor cell lines. After incubation in human plasma, the protein corona was investigated by SDS-PAGE and mass spectrometry analysis. The enzyme nanocarriers generally seemed to favor their biocompatibility, promoting the interaction with dysopsonins. Despite this, PLGA or high degree of PEGylation promoted the adsorption of immunoglobulins with a possible activation of immune response and this effect was probably due to PLGA hydrophobicity and dimensions and to the production of specific antibodies against PEG. In conclusion, the PEGylated MWCNTs at low degree seemed the most biocompatible nanocarrier for adsorbed DAAO, preserving its anticancer activity and forming a bio-corona able to reduce both defensive responses and blood clearance.

Blood D-Amino Acid Oxidase Levels Increased With Cognitive Decline Among People With Mild Cognitive Impairment: A Two-Year Prospective Study.

BackgroundDysregulation of N-methyl-D-aspartate receptor (NMDAR) neurotransmission has been reported to be implicated in the pathogenesis of Alzheimer’s disease (AD). D-amino acid oxidase (DAO), responsible for degradation of NMDAR-related D-amino acids such as D-serine, regulates NMDAR function. A cross-section study found that serum DAO levels were positively related with the severity of cognitive aging among elderly individuals. This 2-year prospective study aimed to explore the role of DAO levels in predicting the outcome of patients with very early-phase AD, such as mild cognitive impairment (MCI).MethodsFifty-one patients with MCI and 21 healthy individuals were recruited. Serum DAO levels and cognitive function, measured by the AD assessment scale-cognitive subscale and the Mini-Mental Status Examination, were monitored every 6 months. We employed multiple regressions to examine the role of DAO concentration in cognitive decline in the 2-year period.ResultsFrom baseline to endpoint (24 months), serum DAO levels increased significantly, and cognitive ability declined according to both cognitive tests in the MCI patients. Among the healthy individuals, DAO concentrations also increased and Mini-Mental Status Examination scores declined; however, AD assessment scale-cognitive subscale scores did not significantly change. Further, DAO levels at both months 12 and 18 were predictive of cognitive impairment at month 24 among the MCI patients.ConclusionsTo our knowledge, this is the first study to demonstrate that blood DAO levels increased with cognitive deterioration among the MCI patients in a prospective manner. If replicated by future studies, blood DAO concentration may be regarded as a biomarker for monitoring cognitive change in the patients with MCI.

AutoDesigner, a De Novo Design Algorithm for Rapidly Exploring Large Chemical Space for Lead Optimization: Application to the Design and Synthesis of d-Amino Acid Oxidase Inhibitors.

The lead optimization stage of a drug discovery program generally involves the design, synthesis, and assaying of hundreds to thousands of compounds. The design phase is usually carried out via traditional medicinal chemistry approaches and/or structure-based drug design (SBDD) when suitable structural information is available. Two of the major limitations of this approach are (1) difficulty in rapidly designing potent molecules that adhere to myriad project criteria, or the multiparameter optimization (MPO) problem, and (2) the relatively small number of molecules explored compared to the vast size of chemical space. To address these limitations, we have developed AutoDesigner, a de novo design algorithm. AutoDesigner employs a cloud-native, multistage search algorithm to carry out successive rounds of chemical space exploration and filtering. Millions to billions of virtual molecules are explored and optimized while adhering to a customizable set of project criteria such as physicochemical properties and potency. Additionally, the algorithm only requires a single ligand with measurable affinity and a putative binding model as a starting point, making it amenable to the early stages of an SBDD project where limited data are available. To assess the effectiveness of AutoDesigner, we applied it to the design of novel inhibitors of d-amino acid oxidase (DAO), a target for the treatment of schizophrenia. AutoDesigner was able to generate and efficiently explore over 1 billion molecules to successfully address a variety of project goals. The compounds generated by AutoDesigner that were synthesized and assayed (1) simultaneously met not only physicochemical criteria, clearance, and central nervous system (CNS) penetration (Kp,uu) cutoffs but also potency thresholds and (2) fully utilize structural data to discover and explore novel interactions and a previously unexplored subpocket in the DAO active site. The reported data demonstrate that AutoDesigner can play a key role in accelerating the discovery of novel, potent chemical matter within the constraints of a given drug discovery lead optimization campaign.

P 13 Application of antipsychotic drugs in the treatment of schizophrenia according to single nucleotide polymorphisms of risk genes

Introduction: Schizophrenia is a chronic mental disease which has a genetic etiology in 80% of the cases. In the last years, about 260 risk genes with a predisposition to schizophrenia have been described, and correlations between single nucleotide polymorphisms (SNPs) of risk genes and the therapeutic efficacy of an antipsychotic treatment/pharmacotherapy resistance have been reported. Methods: In schizophrenia, important risk genes, such as catechol-O-methyl transferase (COMT), monoamine oxidase (MAO A/B), glutamic acid decarboxylase 67 (GAD 67), dysbindin-1 and neuregulin-1 are mentioned. To describe the function of these risk genes, neural networks in the ventral tegmental area, hippocampus and prefrontal cortex are developed. Results: An association between the SNPs of some risk genes and the efficacy of an antipsychotic treatment is reported: SNPs such as rs165599 ( COMT gene), rs1801028 ( D2 receptor gene) and rsSer9 Gly ( D3 receptor gene) are associated with a better antipsychotic treatment efficacy. In this case, a treatment of positive and negative schizophrenic symptoms with risperidone is secured. The rs4680 SNP ( COMT and D2 receptor genes) is associated with antipsychotic-induced dopamine hypersensitivity and pharmacotherapy resistance. The function of the COMT and MAO A/B risk genes is described. In schizophrenia, through a reduced activity in the corresponding enzymes, a decrease in dopamine degradation occurs and hence, via D2 receptors, and dopamine hyperactivity has been observed. The GAD 67 risk gene is linked with GABAergic dysfunction and consequently GABAergic neurons weakly presynaptically inhibit D2 dopaminergic neurons. The D-amino acid oxidase activator (DAOA) risk gene is connected with glutamatergic dysfunction via NMDA receptors. Glutamatergic neurons might exert a weak presynaptic inhibition upon 5-HT2A serotonergic neurons located in the ventral tegmental area and hippocampus. Conclusion: It is important to examine the SNPs of the risk genes involved in schizophrenia to establish a correlation between these SNPs and the efficacy of a determined antipsychotic drug. Thus, schizophrenic patients with a good response to a determined antipsychotic treatment and patients with resistance to this treatment could be well differentiated. The latter patients with pharmacoresistance might be treated with the antipsychotic drug clozapine and an additional treatment with cariprazine, a partial agonist at D2 and D3 receptors.

Coumarin derivatives as inhibitors of d-amino acid oxidase and monoamine oxidase

d-Amino acid oxidase (DAAO) oxidises d-amino acids to ultimately produce the corresponding α-keto acids. The DAAO substrate, d-serine, is a co-agonist at NMDA receptors, while NMDA receptor hypo-function has been implicated in the pathophysiology of schizophrenia. Through the modulation of d-serine levels, the inhibition of DAAO represents a strategy to increase NMDA receptor function, and thus a potential treatment for schizophrenia. Literature reports that 3-hydroxycoumarin is a potent inhibitor of DAAO and represents an ideal lead for the development of novel DAAO inhibitors. Based on this, the present study investigated DAAO inhibition by a series of synthetic and commercially available coumarin derivatives. Due to structural similarity to coumarin, a synthetic series of 3,4-dihydroisoquinolin-1(2H)-one derivatives has also been included in this study. The results show that among 37 compound evaluated, four inhibit porcine kidney DAAO with IC50 < 10 µM. The most potent inhibitors are 3,7-dihydroxycoumarin and 6,7-dihydroxycoumarin with an IC50 values of 0.167 and 0.224 µM, respectively. These values are an improvement on that of the reference DAAO inhibitor, 3-methylpyrazole-5-carboxylic acid (IC50 = 1.88 µM). Coumarin compounds are also known to inhibit the monoamine oxidase (MAO) enzymes, which are well established targets for the treatment of depression and Parkinson’s disease. As DAAO and MAO are flavoenzymes, off-target inhibition may occur. The series were thus evaluated as potential MAO inhibitors, and a number of high potency inhibitors were identified. Seven compounds inhibit the recombinant human MAOs with IC50 < 0.1 µM, with the most potent MAO-A and MAO-B inhibitors exhibiting IC50 values of 0.033 and 0.012 µM, respectively. This is significantly more potent than the reference inhibitors, curcumin, isatin and toloxatone. This study concludes that active DAAO and MAO inhibitors may serve as novel leads for the design of compounds that may find future application in the treatment of neuropsychiatric (e.g. schizophrenia, depression) and neurodegenerative disorders (e.g. Parkinson’s disease).

D-Cysteine Activates Chaperone-Mediated Autophagy in Cerebellar Purkinje Cells via the Generation of Hydrogen Sulfide and Nrf2 Activation.

Chaperone-mediated autophagy (CMA) is a pathway in the autophagy-lysosome protein degradation system. CMA impairment has been implicated to play a role in spinocerebellar ataxia (SCA) pathogenesis. D-cysteine is metabolized by D-amino acid oxidase (DAO), leading to hydrogen sulfide generation in the cerebellum. Although D-cysteine alleviates the disease phenotypes in SCA-model mice, it remains unknown how hydrogen sulfide derived from D-cysteine exerts this effect. In the present study, we investigated the effects of D-cysteine and hydrogen sulfide on CMA activity using a CMA activity marker that we have established. D-cysteine activated CMA in Purkinje cells (PCs) of primary cerebellar cultures where DAO was expressed, while it failed to activate CMA in DAO-deficient AD293 cells. In contrast, Na2S, a hydrogen sulfide donor, activated CMA in both PCs and AD293 cells. Nuclear factor erythroid 2-related factor 2 (Nrf2) is known to be activated by hydrogen sulfide and regulate CMA activity. An Nrf2 inhibitor, ML385, prevented CMA activation triggered by D-cysteine and Na2S. Additionally, long-term treatment with D-cysteine increased the amounts of Nrf2 and LAMP2A, a CMA-related protein, in the mouse cerebellum. These findings suggest that hydrogen sulfide derived from D-cysteine enhances CMA activity via Nrf2 activation.

D-serine metabolism in the medial prefrontal cortex, but not the hippocampus, is involved in AD/HD-like behaviors in SHRSP/Ezo

Attention-deficit/hyperactivity disorder (AD/HD) is a mild neurodevelopmental disorder with inattention, hyperactivity, and impulsivity as its core symptoms. We previously revealed that an AD/HD animal model, juvenile stroke-prone spontaneously hypertensive rats (SHRSP/Ezo) exhibited functional abnormalities in N-methyl-D-aspartate (NMDA) receptors in the prefrontal cortex. D-serine is an endogenous co-ligand that acts on the glycine-binding site of NMDA receptors, which is essential for the physiological activation of NMDA receptors. We herein performed neurochemical and pharmacological behavioral experiments to elucidate dysfunctions in D-serine metabolism (namely, biosynthesis and catabolism) associated to AD/HD.The serine enantiomers ratio (D-serine/D-serine + L-serine, DL ratio) in the medial prefrontal cortex (mPFC) and hippocampus (HIP) was lower in SHRSP/Ezo than in its genetic control. The level of D-amino acid oxidase (DAAO, D-serine degrading enzyme) was higher in the mPFC, and the level of serine racemase (SR, D-serine biosynthetic enzyme), was lower in the HIP in SHRSP/Ezo. Thus, changes in these enzymes may contribute to the lower DL ratio of SHRSP/Ezo. Moreover, a microinjection of a DAAO inhibitor into the mPFC in SHRSP/Ezo increased DL ratio and attenuated AD/HD-like behaviors, such as inattention and hyperactivity, in the Y-maze test. Injection into the HIP also increased the DL ratio, but had no effect on behaviors.These results suggest that AD/HD-like behaviors in SHRSP/Ezo are associated with an abnormal D-serine metabolism underlying NMDA receptor dysfunction in the mPFC. These results will contribute to elucidating the pathogenesis of AD/HD and the development of new treatment strategies for AD/HD.

Free d-Amino Acids in Salivary Gland in Rat.

Simple SummaryParotid, submandibular, and sublingual glands in rat were found to contain high concentrations of d-aspartic acid and low ones of d-serine and d-alanine. In addition to d-amino acid oxidase and d-aspartate oxidase, serine racemase was also detected in all three of these major salivary glands, as were N-methyl-d-aspartic acid receptor subunits NR1 and NR2D, but not NR2A, NR2B, or NR2C.Free d-amino acids, which are enantiomers of l-amino acids, are found in mammals, including humans, and play an important role in a range of physiological functions in the central nervous system and peripheral tissues. Several d-amino acids have been observed in saliva, but their origin and the enzymes involved in their metabolism and catabolism remain to be clarified. In the present study, large amounts of d-aspartic acid and small amounts of d-serine and d-alanine were detected in all three major salivary glands in rat. No other d-enantiomers were detected. Protein expression of d-amino acid oxidase and d-aspartate oxidase, the enzymes responsible for the oxidative deamination of neutral and dicarboxylic d-amino acids, respectively, were detected in all three types of salivary gland. Furthermore, protein expression of the d-serine metabolic enzyme, serine racemase, in parotid glands amounted to approximately 40% of that observed in the cerebral cortex. The N-methyl-d-aspartic acid subunit proteins NR1 and NR2D were detected in all three major salivary glands. The results of the present study suggest that d-amino acids play a physiological role in a range of endocrine and exocrine function in salivary glands.

Investigation of Human in vivo Metabolism of SEP-227900 Using the Samples from First-in-Human Study by LC-HRMS/UV and NMR.

The study aims to explore the human in vivo metabolism of SEP-227900 (4H-furo[3, 2-b] pyrrole-carboxylic acid, m.w 151.03), a D-amino-acid oxidase (DAAO) inhibitor, by using plasma and urine samples from first-in-human study. The human plasma and urine samples were from a single dose cohort that consisted of 9 healthy male volunteers each received an 80- mg dose of SEP-227900 orally. The pooled pre-dose urine and the pooled 0-24 h urine sample were created across 9 subjects by equal volume. Plasma samples were pooled by equal volume across 9 subjects to obtain 0-12 h plasma for metabolite searching, and also pooled by timepoints across 9 subjects to obtain 0.5, 5, and 12-h plasma for semi-quantitation. The plasma was de-proteinized by acetonitrile (1:3 v/v plasma-acetonitrile), then the supernatant was dried down, reconstituted, and injected for LC-HRMS/UV analysis. The urine sample was just simply centrifuged before analysis. LC-HRMS/UV was utilized to search predictable and unknown metabolites and estimate their relative abundances. Accurate mass measurement by Orbitrap-MS and MS/MS was used for metabolite identification. Chromatographic separation was achieved on a MACMOD AQ C8 column (250 × 4.6 mm, 5-μm) with a gradient mobile phase (A: 10 mM NH4Ac; B: acetonitrile; flowrate: 0.700 ml/min) for a total run-time of 65 min. The definite position in the molecule for the glucuronidation metabolism was characterized by the detected migration phenomenon, methylation with diazomethane (CH2N2), and NMR. Unchanged parent drug and four metabolite peaks were detected in humans: M1 was a mono-oxidative metabolite of SEP-227900; M2 was a glucuronide conjugate of SEP-227900; M3 was a glycine conjugate of SEP-227900; M4 was a glycine conjugate of M1. The specific position of the oxidation in M1 solely based on the mass spectral (MS and MS/MS) data was not identified. However, for the major metabolite M2, the acyl glucuronidation was unambiguously determined through multiple pieces of experimental evidence such as the observation of a migration pattern, mono-methylation by diazomethane, and NMR measurement. This determination is of significance related to the safety evaluation of investigational new drug development. The glycine conjugate of SEP-227900, i.e., M3, was found to be the most abundant metabolite in human urine (approximately 3-fold higher level than the glucuronide level). All together (mainly glycine-conjugate and glucuronide), it resulted in greater than 80% of the dosed amount in urine excretion (a separate measurement showed 23% of the dosed amount in urine excretion as the glucuronide). Four metabolites were found in humans: SEP-227900-glycine conjugate, SEP- 227900-glucuronide, mono-oxidative metabolite, and its consequent glycine conjugate. The glucuronide metabolite was identified as acyl glucuronide. Greater than 80% of the dosed amount of SEP-227900 was excreted in the urine, mainly in the forms of glycine- and glucuronide- conjugates.

Treatment with Polyethylene Glycol–Conjugated Fungal d-Amino Acid Oxidase Reduces Lung Inflammation in a Mouse Model of Chronic Granulomatous Disease

Chronic granulomatous disease (CGD) is a primary immunodeficiency wherein phagocytes are unable to produce reactive oxygen species (ROS) owing to a defect in the nicotinamide adenine dinucleotide phosphate oxidase (NADPH) complex. Patients with CGD experience bacterial and fungal infections and excessive inflammatory disorders. Bone marrow transplantation and gene therapy are theoretically curative; however, residual pathogenic components cause inflammation and/or organic damage in patients. Moreover, antibiotic treatments may not help in preventing excessive inflammation due to the residual presence of fungal cell wall β-glucan. Thus, better treatment strategies against CGD are urgently required. Polyethylene glycol–conjugated recombinant porcine d-amino acid oxidase (PEG-pDAO) supplies ROS to defective NADPH oxidase in neutrophils of patients with CGD, following which the neutrophils regain bactericidal activity in vitro. In this study, we employed an in vivo nonviable Candida albicans (nCA)–induced lung inflammation model of gp91-phox knockout CGD mice and supplied novel PEG conjugates of Fusarium spp. d-amino acid oxidase (PEG-fDAO), as it exhibits higher enzyme activity than PEG-pDAO. The body weight, lung weight, and lung pathology were evaluated using three experimental strategies with the in vivo lung inflammation model to test the efficacy of the ROS-generating enzyme replacement therapy with PEG-fDAO. The lung weight and pathological findings suggest the condition was ameliorated by administration PEG-fDAO, followed by intraperitoneal injection of d-phenylalanine or d-proline. Although a more precise protocol is essential, these data reveal the targeted delivery of PEG-fDAO to the nCA-induced inflammation site and show that PEG-fDAO can be used to treat inflammation in CGD in vivo.

Combining a Genetically Engineered Oxidase with Hydrogen-Bonded Organic Frameworks (HOFs) for Highly Efficient Biocomposites.

Enzymes incorporated into hydrogen‐bonded organic frameworks (HOFs) via bottom‐up synthesis are promising biocomposites for applications in catalysis and sensing. Here, we explored synthetic incorporation of d‐amino acid oxidase (DAAO) with the metal‐free tetraamidine/tetracarboxylate‐based BioHOF‐1 in water. N‐terminal enzyme fusion with the positively charged module Zbasic2 strongly boosted the loading (2.5‐fold; ≈500 mg enzyme gmaterial −1) and the specific activity (6.5‐fold; 23 U mg−1). The DAAO@BioHOF‐1 composites showed superior activity with respect to every reported carrier for the same enzyme and excellent stability during catalyst recycling. Further, extension to other enzymes, including cytochrome P450 BM3 (used in the production of high‐value oxyfunctionalized compounds), points to the versatility of genetic engineering as a strategy for the preparation of biohybrid systems with unprecedented properties.

Reactive Oxygen Species Differentially Modulate the Metabolic and Transcriptomic Response of Endothelial Cells.

Reactive oxygen species (ROS) are important mediators of both physiological and pathophysiological signal transduction in the cardiovascular system. The effects of ROS on cellular processes depend on the concentration, localization, and duration of exposure. Cellular stress response mechanisms have evolved to mitigate the negative effects of acute oxidative stress. In this study, we investigate the short-term and long-term metabolic and transcriptomic response of human umbilical vein endothelial cells (HUVEC) to different types and concentrations of ROS. To generate intracellular H2O2, we utilized a lentiviral chemogenetic approach for overexpression of human D-amino acid oxidase (DAO). DAO converts D-amino acids into their corresponding imino acids and H2O2. HUVEC stably overexpressing DAO (DAO-HUVEC) were exposed to D-alanine (3 mM), exogenous H2O2 (10 µM or 300 µM), or menadione (5 µM) for various timepoints and subjected to global untargeted metabolomics (LC-MS/MS) and RNAseq by MACE (Massive analysis of cDNA ends). A total of 300 µM H2O2 led to pronounced changes on both the metabolic and transcriptomic level. In particular, metabolites linked to redox homeostasis, energy-generating pathways, and nucleotide metabolism were significantly altered. Furthermore, 300 µM H2O2 affected genes related to the p53 pathway and cell cycle. In comparison, the effects of menadione and DAO-derived H2O2 mainly occurred at gene expression level. Collectively, all types of ROS led to subtle changes in the expression of ribosomal genes. Our results show that different types and concentration of ROS lead to a different metabolic and transcriptomic response in endothelial cells.

Efficacy and safety of add-on sodium benzoate, a D-amino acid oxidase inhibitor, in treatment of schizophrenia: A systematic review and meta-analysis

The role of sodium benzoate, an NMDA receptor enhancer, in schizophrenia has been evaluated in a few clinical trials, but results are contradictory and inconclusive. The present meta-analysis has evaluated the efficacy and safety of add-on sodium benzoate for the treatment of schizophrenia. After performing a literature search on MEDLINE/PubMed, Scopus, Cochrane databases and International Clinical Trial Registry Platform, reviewers assessed eligibility and extracted data from four relevant articles. PRISMA guidelines were followed in the selection, analysis, and reporting of findings. The random-effect model was used to estimate effect size. Quality assessment was done using the risk of bias assessment tool, and sensitivity analysis was done in case of high heterogeneity. Add-on sodium benzoate can improve positive symptoms of schizophrenia significantly (MD: -1.87; 95%CI: -3.25 to -0.48; p=0.008) but had no significant favourable effect on negative symptoms (p=0.84), general psychopathology (p=0.49), and total PANSS score (p=0.19) over the control. There was no significant improvement in GAF (p=0.43), CGI (p=0.58), cognitive function (p=0.46) and quality of life (p=0.73). Extrapyramidal symptoms were significantly higher (MD: 0.39; 95% CI:0.19-0.60; p=0.0002) in the sodium benzoate group in comparison to the control group; however, there was no significant difference in respect to other adverse events. Sodium benzoate can improve the positive symptoms of schizophrenia without any beneficial effect on other symptomatology, cognition, quality of life and functioning. Further studies are needed to evaluate long-term efficacy, safety and use in specific subgroups of patients.

Effects of Sodium Benzoate, a D-Amino Acid Oxidase Inhibitor, on Perceived Stress and Cognitive Function Among Patients With Late-Life Depression: A Randomized, Double-Blind, Sertraline- and Placebo-Controlled Trial.

BackgroundCompared with adults with depression in the general population, elderly depressive patients are prone to poor treatment response, more side effects, and early withdrawal with current antidepressants (which principally modulate monoamines). Whether N-methyl-D-aspartate receptor enhancement can benefit treatment of late-life depression deserves study. This study aims to compare sodium benzoate (a D-amino acid oxidase inhibitor and an indirect N-methyl-D-aspartate receptor enhancer), sertraline (a selective serotonin reuptake inhibitor), and placebo in the treatment of late-life depression.MethodsIn this randomized, double-blind trial, 117 patients with major depressive disorder aged 55 years or older received 8-week treatment of 250–1500 mg/d of sodium benzoate, 25–150 mg/d of sertraline, or placebo in 2 medical centers. The primary outcome measures were Hamilton Depression Rating Scale and Perceived Stress Scale scores.ResultsThree treatments similarly decreased clinicians-rated Hamilton Depression Rating Scale scores. Compared with placebo, sodium benzoate but not sertraline substantially improved Perceived Stress Scale scores and cognitive function. Sertraline, but not benzoate, significantly reduced self-report Geriatric Depression Scale scores. Benzoate and placebo showed similar safety profiles, while sertraline was more likely to raise low-density lipoprotein than benzoate and placebo. Benzoate-treated patients were less likely to drop out than sertraline or placebo recipients.ConclusionsSertraline can reduce subjective depressive symptoms, while benzoate can decrease perceived stress, improve cognitive function, and enhance treatment adherence in late-life depression patients. The results show promise for D-amino acid oxidase inhibition as a novel approach for perceived stress and cognitive decline among patients with late-life depression.Trial RegistrationClinicalTrials.gov Identifier: NCT03414931. Registered January 2016.