Background: DAB389IL-2 (Denileukin diftitox) as an immunotoxin is a targeted pharmaceutical protein and is the first immunotoxin approved by FDA. It is used for the treatment of various kinds of cancer such as CTCL lymphoma, melanoma, and Leukemia but among all of these, treatment of CTCL has special importance. DAB389IL-2 consists of two distinct parts; the catalytic domain of Diphtheria Toxin (DT) that genetically fused to the whole IL-2. Deamidation is the most important reaction for chemical instability of proteins occurs during manufacture and storage. Deamidation of asparagine residues occurs at a higher rate than glutamine residues. The structure of proteins, temperature and pH are the most important factors that influence the rate of deamidation.

Methods: Since there is not any information about deamidation of DAB389IL-2, we studied in silico deamidation by Molecular Dynamic (MD) simulations using GROMACS software. The 3D model of fusion protein DAB389IL-2 was used as a template for deamidation. Then, the stability of deamidated and native form of the drug was calculated.

Results: The results of MD simulations were showed that the deamidated form of DAB389IL-2 is more unstable than the normal form. Also, deamidation was carried by incubating DAB389IL-2, 0.3 mg/ml in ammonium hydrogen carbonate for 24 h at 37o C in order to in vitro experiment.

Conclusion: The results of in vitro experiment were confirmed outcomes of in silico study. In silico and in vitro experiments were demonstrated that DAB389IL-2 is unstable in deamidated form.

背景：DAB389IL-2（Denileukin diftitox）作为一种免疫毒素是一种靶向药物蛋白，是FDA批准的首种免疫毒素。它用于治疗各种癌症，例如CTCL淋巴瘤，黑色素瘤和白血病，但是在所有这些癌症中，CTCL的治疗特别重要。DAB389IL-2由两个不同的部分组成；在遗传上与整个IL-2融合的白喉毒素（DT）的催化结构域。脱酰胺作用是最重要的反应，因为蛋白质在制造和储存过程中会发生化学不稳定。天冬酰胺残基的脱酰胺作用发生的速度高于谷氨酰胺残基。蛋白质的结构，温度和pH是影响脱酰胺速率的最重要因素。

方法：由于没有有关DAB389IL-2脱酰胺的信息，我们使用GROMACS软件通过分子动力学（MD）模拟对计算机脱酰胺进行了研究。融合蛋白DAB389IL-2的3D模型用作脱酰胺的模板。然后，计算了脱酰胺和天然形式药物的稳定性。

结果：MD模拟的结果表明，DAB389IL-2的脱酰胺化形式比正常形式更不稳定。另外，通过在碳酸氢铵中于0.3 oC下孵育0.3 mg / ml的DAB389IL-2进行脱酰胺作用，以进行体外实验。

结论：体外实验结果证实了计算机模拟研究的结果。在计算机和体外实验中证明，DAB389IL-2以脱酰胺基形式不稳定。