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.

中文翻译：

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PEG-DAAO共轭物：一种通过蛋白质工程优化的有前途的癌症治疗工具。

黄酮酶D-氨基酸氧化酶（DAAO）代表癌症酶前药治疗的潜在好选择，因为它以D-氨基酸为底物，体内低浓度存在的化合物产生H2O2，并且可以安全地给药以调节H2O2的产生。我们通过以下方法优化了治疗的细胞毒性：i）使用在低O2和D-丙氨酸浓度（mDAAO）下有活性的有效酶变体；ii）通过聚乙二醇化改善mDAAO的稳定性和半衰期以及增强的渗透性和保留效果；iii）通过血红素加氧酶-1抑制剂（ZnPP）抑制抗氧化剂细胞系统。极少量的PEG-mDAAO（10 mU，50 ng的酶）会诱导各种肿瘤细胞系的细胞毒性。尤其，