An antibody-based enzymatic therapy for cancer treatment: The selective localization of D-amino acid oxidase to EDA fibronectin.

In order to generate an antibody directed enzyme prodrug therapy, here we designed a chimeric protein by fusing the F8 antibody that recognizes the EDA of fibronectin (expressed on the tumor neovasculature) and an evolved variant of the ROS-generating enzyme D-amino acid oxidase (DAAO). The F8(scFv)-DAAO-Q144R recombinant protein is expressed by both CHO-S and E. coli cells. The F8(scFv)-DAAO-Q144R from E. coli cells is fully soluble, shows a high specific activity, is more thermostable in blood than the native DAAO, possesses a binding affinity for EDA well suited for efficient tumor accumulation, and localizes in tumor tissues. Notably, the F8(scFv)-DAAO-Q144R conjugate generates a stronger cytotoxicity to tumor cells than the native enzyme, especially when an inhibitor of heme oxygenase-1 (HO-1) is used, making it a promising candidate for a selective antitumor oxidative therapy controlled by the substrate addition, in the so called "activity on demand", thus sparing normal tissue from damage.

CLytA-DAAO, Free and Immobilized in Magnetic Nanoparticles, induces Cell Death in Human Cancer Cells.

D-amino acid oxidase (DAAO) catalyzes the oxidation of D-amino acids generating hydrogen peroxide, a potential producer of reactive oxygen species. In this study, we used a CLytA-DAAO chimera, both free and bound to magnetic nanoparticles, against colon carcinoma, pancreatic adenocarcinoma, and glioblastoma cell lines. We found that the enzyme induces cell death in most of the cell lines tested and its efficiency increases significantly when it is immobilized in nanoparticles. We also tested this enzyme therapy in non-tumor cells, and we found that there is not cell death induction, or it is significantly lower than in tumor cells. The mechanism triggering cell death is apparently a classical apoptosis pathway in the glioblastoma cell lines, while in colon and pancreatic carcinoma cell lines, CLytA-DAAO-induced cell death is a necrosis. Our results constitute a proof of concept that an enzymatic therapy, based on magnetic nanoparticles-delivering CLytA-DAAO, could constitute a useful therapy against cancer and besides it could be used as an enhancer of other treatments such as epigenetic therapy, radiotherapy, and treatments based on DNA repair.

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.

Immobilization of enzymes using a multifunctional fusion polypeptide.

OBJECTIVE: To design a fusion polypeptide combining functions of self-assembly and purification for immobilizing enzymes. RESULTS: A collagen-like polypeptide (CLP) was fused to an elastin-like polypeptide (ELP) through genetic engineering. CLP-ELP was separately fused to superoxide dismutase (SOD) and D-amino acid oxidase (DAAO). The recombinant enzymes were purified with using reversible phase transition. The interfering effect of H2O2 on the secondary structures of the recombinant enzymes was significantly reduced. The stability of the recombinant enzymes against denaturing by urea was improved. SOD-CLP-ELP exhibited a proteinaceous microporous network, and DAAO-CLP-ELP exhibited micro-clusters. The superoxide anion (•O2-) scavenging ability of SOD-CLP-ELP was 1.5 times that of SOD, and the catalytic efficiency of DAAO-CLP-ELP was 1.7 times that of DAAO. CONCLUSIONS: The advantages of the CLP-ELP-fused enzymes have been demonstrated and CLP-ELP can be used to immobilize other enzymes/proteins.

High-Throughput Screening Strategy Identifies Allosteric, Covalent Human D-Amino Acid Oxidase Inhibitor.

Genome-wide association studies have linked polymorphisms in the gene G72 to schizophrenia risk in several human populations. Although controversial, biochemical experiments have suggested that the mechanistic link of G72 to schizophrenia is due to the G72 protein product, pLG72, exerting a regulatory effect on human D-amino acid oxidase (hDAAO) activity. In an effort to identify hDAAO inhibitors of novel mechanism of action, we designed a pLG72-directed hDAAO activity assay suitable for high-throughput screening (HTS). During assay development, we confirmed that pLG72 was an inhibitor of hDAAO. Thus, our assay employed an IC20 pLG72 concentration that was high enough to allow dynamic pLG72-hDAAO complexes to form but with sufficient remaining hDAAO activity to measure during an HTS. After conducting an approximately 150,000-compound HTS, we further characterized a class of compound hits that were less potent hDAAO inhibitors when pLG72 was present. Focusing primarily on compound 2: [2-(2,5-dimethylphenyl)-6-fluorobenzo[d]isothiazol-3(2H)-on], we demonstrated that these compounds inhibited hDAAO via an allosteric, covalent mechanism. Although there is significant interest in the therapeutic potential of compound 2: and its analogues, their sensitivity to reducing agents and their capacity to bind cysteines covalently would need to be addressed during therapeutic drug development.

Enhancement of the solubility and stability of D-amino acid oxidase by fusion to an elastin like polypeptide.

An elastin-like polypeptide (ELP) was fused to D-amino acid oxidases (DAAO). ELP-DAAO exhibited a better solubility in aqueous solutions than DAAO, and its enzymatic activity is about 1.6 times that of DAAO. The stability of the proteins was investigated by interacting with urea at various concentrations. The circular dichroism and fluorescence spectra were measured. The results demonstrated that that ELP-DAAO exhibited a much better stability than DAAO, and ELP-DAAO has retained the α-helix content with a high percentage even at a high urea concentration. The results of this work have demonstrated that the ELP tag can be utilized to purify DAAO, in the meantime the solubility and stability of the enzyme are improved.

Structure-function relationships in human d-amino acid oxidase variants corresponding to known SNPs.

In the brain, d-amino acid oxidase plays a key role in modulating the N-methyl-d-aspartate receptor (NMDAR) activation state, catalyzing the stereospecific degradation of the coagonist d-serine. A relationship between d-serine signaling deregulation, NMDAR dysfunction, and CNS diseases is presumed. Notably, the R199W substitution in human DAAO (hDAAO) was associated with familial amyotrophic lateral sclerosis (ALS), and further coding substitutions, i.e., R199Q and W209R, were also deposited in the single nucleotide polymorphism database. Here, we investigated the biochemical properties of these different hDAAO variants. The W209R hDAAO variant shows an improved d-serine degradation ability (higher activity and affinity for the cofactor FAD) and produces a greater decrease in cellular d/(d+l) serine ratio than the wild-type counterpart when expressed in U87 cells. The production of H2O2 as result of excessive d-serine degradation by this hDAAO variant may represent the factor affecting cell viability after stable transfection. The R199W/Q substitution in hDAAO altered the protein conformation and enzymatic activity was lost under conditions resembling the cellular ones: this resulted in an abnormal increase in cellular d-serine levels. Altogether, these results indicate that substitutions that affect hDAAO functionality directly impact on d-serine cellular levels (at least in the model cell system used). The pathological effect of the expression of the R199W hDAAO, as observed in familial ALS, originates from both protein instability and a decrease in kinetic efficiency: the increase in synaptic d-serine may be mainly responsible for the neurotoxic effect. This information is expected to drive future targeted treatments.

D-amino acid oxidase-nanoparticle system: a potential novel approach for cancer enzymatic therapy.

AIM: The authors propose a new magnetic nanoparticle-enzyme system for cancer therapy capable of targeting the enzyme and consequently decreasing the adverse effects, meanwhile improving the patient's life quality. MATERIALS & METHODS: The authors have functionalized Fe3O4 nanoparticles with 3-amino-propyltriethoxysilane (APTES) and conjugated it to yeast D-amino acid oxidase (DAAO) by coupling this with glutaraldehyde. RESULTS & CONCLUSION: The authors have tested the Fe3O4-APTES-DAAO system on three tumor cell lines. Exposed cells show, at the electron microscope level, nanoparticles on the surface of the plasma membrane and inside endocytic vesicles. Fe3O4-APTES-DAAO caused a substantial decrease of cell viability greatly augmented when D-alanine, a DAAO substrate, was added. Fe3O4-APTES-DAAO was demonstrated to be more effective than free DAAO, confirming the validity of the system in cancer therapy.

Fabrication of an amperometric D-amino acid biosensor based on nickel hexacyanoferrate polypyrrole hybrid film deposited on glassy carbon electrode.

A nickel hexacyanoferrate polypyrrole film was synthesized through an electrochemical two-step methodology leading to a very stable and homogenous robust hybrid film. A highly sensitive, specific and rapid amperometric D-amino acid biosensor was constructed by immobilizing D-amino acid oxidase on this film deposited over the surface of a glassy carbon electrode. The modified electrode was characterized by scanning electron microscopy, electrochemical impedance spectroscopy and Fourier transform infrared spectrophotometry. The biosensor showed optimum response within 1 s, when operated at 50 mV s(-1) in 0.01 M Tris HCl buffer, pH 7.0 at 30 °C. The biosensor exhibited excellent sensitivity with a detection limit of 1.5 µM (S/N = 3) for D-amino acids and wider linear range, 20-500 µM. Analytical recovery of added D-alanine (5 and 10 mM) in serum samples was 98.00 and 98.80 %, respectively. Within-batch and between-batch coefficients of variation in serum samples were 1.36 and 2.77 %, respectively. The enzyme electrode was used more than 50 times over 2 months, when stored at 4 °C. The proposed modified electrode exhibited sufficient mechanical and electrochemical stability and high sensitivity compared to earlier electrochemical D-amino acid biosensors. Interference by ascorbic acid and uric acid, the main interfering species in the biological samples, was negligible.

Characterization of human DAAO variants potentially related to an increased risk of schizophrenia.

Considering the key role of d-serine in N-methyl-d-aspartate receptor-mediated neurotransmission, it is highly relevant to define the role that enzymes play in d-serine synthesis and degradation. In particular, the details of regulation of the d-serine catabolic human enzyme d-amino acid oxidase (hDAAO) are unknown although different lines of evidence have shown it to be involved in schizophrenia susceptibility. Here we investigated the effect of three single nucleotide polymorphisms and known mutations in hDAAO, i.e., D31H, R279A, and G331V. A very low amount of soluble G331V hDAAO is produced in E. coli cells: the recombinant variant enzyme is fully active. Human U87 glioblastoma cells transiently transfected for G331V hDAAO show a low viability, a significant amount of protein aggregates, and augmented apoptosis. The recombinant D31H and R279A hDAAO variants do not show alterations in tertiary and quaternary structures, thermal stability, binding affinity for inhibitors, and the modulator pLG72, whereas the kinetic efficiency and the affinity for d-serine and for FAD were higher than for the wild-type enzyme. While these effects for the substitution at position 31 cannot be structurally explained, the R279A mutation might affect the hDAAO FAD-binding affinity by altering the "structurally ambivalent" peptide V47-L51. In agreement with the observed increased activity, expression of D31H and R279A hDAAO variants in U87 cells produces a higher decrease in cellular d/(d+l) serine ratio than the wild-type counterpart. In vivo, these substitutions could affect cellular d-serine concentration and its release at synapsis and thus might be relevant for schizophrenia susceptibility.

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.

Characterization of a yeast D-amino acid oxidase microbiosensor for D-serine detection in the central nervous system.

d-Serine is an endogenous ligand for N-methyl-d-aspartate (NMDA) receptors, and alterations in its concentration have been related to several brain disorders, especially schizophrenia. It is therefore an important target neuromodulator for the pharmaceutical industry. To monitor d-serine levels in vivo, we have developed a microbiosensor based on cylindrical platinum microelectrodes, covered with a membrane of poly-m-phenylenediamine (PPD) and a layer of immobilized d-amino acid oxidase from the yeast Rhodotorula gracilis (RgDAAO). By detecting the hydrogen peroxide produced by enzymatic degradation of d-serine, this microbiosensor shows a detection limit of 16 nM and a mean response time of 2 s. Interferences by ascorbic acid, uric acid, l-cysteine, and by biogenic amines and their metabolites are rejected at more than 97% by the PPD layer. Although several d-amino acids are potential substrates for RgDAAO, d-serine was the only endogenous substrate present in sufficient concentration to be detected by our microbiosensor in the central nervous system. When implanted in the cortex of anesthetized rats, this microbiosensor detected the increase in concentration of d-serine resulting from its diffusion across the blood-brain barrier after an intraperitoneal injection. This new device will make it possible to investigate in vivo the variations in d-serine concentrations occurring under normal and pathological conditions and to assess the pharmacological potency of new drugs designed to impact d-serine metabolism.

Stability and stabilization of D-amino acid oxidase from the yeast Trigonopsis variabilis.

The use of DAO (D-amino acid oxidase) for the conversion of cephalosporin C has provided a significant case for the successful implementation of an O(2)-dependent biocatalyst on an industrial scale. Improvement of the operational stability of the immobilized oxidase is, however, an important goal of ongoing process optimization. We have examined DAO from the yeast Trigonopsis variabilis with the aim of developing a rational basis for the stabilization of the enzyme activity at elevated temperature and under conditions of substrate turnover. Loss of activity in the resting enzyme can occur via different paths of denaturation. Partial thermal unfolding and release of the FAD cofactor, kinetically coupled with aggregation, contribute to the overall inactivation rate of the oxidase at 50 degrees C. Oxidation of Cys(108) into a stable cysteine sulfinic acid causes both decreased activity and stability of the enzyme. Strategies to counteract each of the denaturation steps in DAO are discussed. Fusion to a pull-down domain is a novel approach to produce DAO as protein-based insoluble particles that display high enzymatic activity per unit mass of catalyst.

Selective modification of surface-exposed thiol groups in Trigonopsis variabilis D-amino acid oxidase using poly(ethylene glycol) maleimide and its effect on activity and stability of the enzyme.

Covalent modification of purified Trigonopsis variabilis D-amino acid oxidase using maleimide-activated poly(ethylene glycol) 5000 yielded a stable bioconjugate in which three surface-exposed cysteine side chains were selectively derivatized. Compared with the native enzyme, the PEGylated variant displayed substantially (approximately 3.3-fold) slowed dissociation rate of FAD cofactor at 50 degrees C, and this caused a twofold thermostabilization of the enzyme activity. The stability under reaction conditions at 30 degrees C was also markedly enhanced in the PEG-oxidase conjugate. PEGylation did not affect steady-state kinetic parameters for oxidative deamination of D-methionine when 2,6-dichloroindophenol replaced dioxygen as the cosubstrate while it caused a ninefold decrease in substrate catalytic efficiency for the dioxygen-dependent reaction.

Thermal inactivation of D-amino acid oxidase from Trigonopsis variabilis occurs via three parallel paths of irreversible denaturation.

Trigonopsis variabilis D-amino acid oxidase (TvDAO) is a long-known flavoenzyme whose most important biocatalytic application is currently the industrial production of 7-amino-cephalosporanic acid (7-ACA) from cephalosporin C. Lacking mechanistic foundation, rational stabilization of TvDAO for improved process performance remains a problem. We report on results of thermal denaturation studies at 50 degrees C in which two purified TvDAO forms were compared: the native enzyme, and a site-specifically oxidized protein variant that had the side chain of cysteine108 converted into a sulfinic acid and lost 75% of original specific activity. Although inactivation time courses for both enzymes are fairly well described by simple single-exponential decays, the underlying denaturation mechanisms are shown by experiments and modeling to be complex. One main path leading to inactivation is FAD release, a process whose net rate is determined by the reverse association rate constant (k), which is 25-fold lower in the oxidized form of TvDAO. Cofactor dissociation is kinetically coupled to aggregation and can be blocked completely by the addition of free FAD. Aggregation is markedly attenuated in the less stable Cys108-SO(2)H-containing enzyme, suggesting that it is a step accompanying but not causing the inactivation. A second parallel path, characterized by a k-value of 0.26/h that is not dependent on protein concentration and identical for both enzymes, likely reflects thermal unfolding reactions. A third, however, slow process is the conversion of the native enzyme into the oxidized form (k < 0.03/h). The results fully explain the different stabilities of native and oxidized TvDAO and provide an inactivation mechanism-based tool for the stabilization of the soluble oxidase.

Tumor-targeted delivery of polyethylene glycol-conjugated D-amino acid oxidase for antitumor therapy via enzymatic generation of hydrogen peroxide.

Hydrogen peroxide (H(2)O(2)) is a strong oxidant that induces apoptosis of tumor cells in vitro. Here, we investigated the antitumor activity of an H(2)O(2)-generating enzyme, D-amino acid oxidase (DAO), and its conjugate with polyethylene glycol (PEG; PEG-DAO). Compared with DAO, PEG-DAO showed improved pharmacokinetic parameters in mice after i.v. injection. PEG-DAO administered i.v. accumulated selectively in tumor tissue with insignificant accumulation in normal organs and tissues. To generate cytotoxic H(2)O(2) at the tumor site, PEG-DAO was first administrated i.v. to tumor-bearing mice. After an adequate lag time, the substrate of DAO, D-proline, was injected i.p. This treatment resulted in significant suppression of tumor growth compared with tumor growth in control animals (not given treatment; P < 0.001). Similar treatment with native DAO showed no effect under the same conditions. Oxidative metabolites were significantly increased in solid tumors by administration of PEG-DAO followed by D-proline (P < 0.002, compared with the group receiving no treatment), as evidenced by thiobarbituric acid-reactive substance assay. This treatment did not affect results from the metabolites in the liver and kidney. These findings suggest that tumor-targeted delivery of DAO is accomplished by using pegylated enzyme and thereby taking advantage of the enhanced permeability and retention effect in solid tumor. PEG-DAO thus delivered together with D-proline produces remarkable antitumor activity via extensive generation of H(2)O(2).

Limited proteolysis and site-directed mutagenesis reveal the origin of microheterogeneity in Rhodotorula gracilis D-amino acid oxidase.

When analysed by isoelectric focusing, D-amino acid oxidase from the yeast Rhodotorula gracilis normally consists of three molecular isoforms (pI 7.8, 7.4 and 7.2, respectively) all with the same N-terminal sequence. However, only a single band of pI 7.8 is detected with the recombinant wild-type protein expressed in E. coli. To determine whether the molecular basis of this heterogeneity is due to proteolysed forms of the protein, we treated R. gracilis D-amino acid oxidase with various proteases. Limited proteolysis by chymotrypsin and thermolysin produced truncated and nicked monomeric holoenzymes containing two polypeptides of approximately 34 kDa (Met1-Leu312) and one of approximately 5 kDa (Ala319-Arg364 with chymotrypsin or Ala319-Ala362 with thermolysin). On the other hand, treatment with endoproteinase Glu-C gave a dimeric holoenzyme lacking the C-terminal SKL tripeptide. This cleavage of Glu365-Ser366 peptide bond caused the disappearance of the three isoelectric bands and a single homogeneous band (pI 7.2) appeared. To study this protein form, we used site-directed mutagenesis to produce a mutant form of R. gracilis D-amino acid oxidase lacking the SKL C-terminal tripeptide (which is the targeting sequence PTS1 for peroxisomal proteins). As expected, the SKL-deleted mutant gave a single band (pI 7.2) in isoelectric focusing. The three-band pattern of native yeast enzyme was generated by in vitro experiments using an equimolar mixture of the wild-type (pI 7.8) and the SKL-deleted recombinant (pI 7.2) DAAOs. The microheterogeneity of yeast DAAO thus stems from the association of two polypeptide chains differing in the C-terminal tripeptide, giving three different holoenzyme dimers.

Identification of a reactive cysteine in the flavin-binding domain of Rhodotorula gracilis D-amino acid oxidase.

The holoenzyme form of Rhodotorula gracilis D-amino acid oxidase, an 80-kDa homodimer, reacted only to a limited extent with general thiol reagents (2,2'-dithiodipyridine, 5,5'-dithiobis(2-nitrobenzoic acid), and N-[7-(dimethylamino)-4-methylcoumarinyl]maleimide) (60% residual activity), whereas the monomeric apoprotein was completely inactivated and denatured by these reagents. To investigate the presence of thiol residue(s) in the active site of the enzyme, the apoprotein was reconstituted with the 8-(methylsulfonyl)-FAD chemical-affinity probe. Competitive inhibition between this analogue and FAD for apoprotein binding was observed. The covalent attachment of the flavin analogue to the apoprotein was complete after approximately 20 h of incubation and the flavinylated enzyme, containing 8-(cysteinyl)-FAD, was monomeric and inactive. After HPLC isolation of the flavin-labeled tryptic peptides, Cys208 was identified as the only cysteine to react with the FAD analogue. These results show that a single cysteine of R. gracilis D-amino acid oxidase reacts with the flavin analogue and that this is located near or at the FAD-binding domain.

Studies on the structural and functional aspects of Rhodotorula gracilis D-amino acid oxidase by limited trypsinolysis.

The structure-function relationships of purified Rhodotorula gracilis D-amino acid oxidase (in its holo-, apo- and holo-enzyme-benzoate complex forms) was analysed by digestion with trypsin. In all cases trypsin cleaves this 80 kDa dimeric enzyme at the C-terminal region, since the peptide bonds sensitive to proteinase attack are clustered in this region. Digestion of native enzyme with trypsin produced a nicked and truncated form of 38.3 kDa containing two polypeptides of 34 and 5 kDa starting from Met1 and Ala319 respectively, and with detachment of the Thr306-Arg318 and Glu365-Leu368 peptides. Our results show that this 'core', folded into a compact structure, is catalytically competent. The acquisition of this nicked form was marked by a shift from a dimeric to a monomeric active enzyme, a result never previously obtained. The deleted sequences, Thr306-Arg318 and Glu365-Leu368, are essential for the monomer-monomer interaction, and, in particular, the region encompassing Thr306-Arg318 should play an essential role in the dimerization process. interestingly, the Ser308-Lys321 sequence present in the lost peptide corresponds to a sequence not present in other known D-amino acid oxidases [Faotto, Pollegioni, Ceciliani, Ronchi and Pilone (1995) Biotechnol. Lett. 17, 193-198]. A role of the cleaved-off region for the thermostabilization of the enzyme is also discussed.