Background: Cell-Penetrating Peptides (CPPs), a family of short peptides, are broadly used as the carrier in the delivery of drugs and different therapeutic agents. Thanks to the existence of valuable databases, computational screening of the experimentally validated CPPs can help the researchers to select more effective CPPs for the intercellular delivery of therapeutic proteins. Arginine deiminase of Mycoplasma hominis, an arginine-degrading enzyme, is currently in the clinical trial for treating several arginine auxotrophic cancers. However, some tumor cells have developed resistance to ADI treatment. The ADI resistance arises from the over-expression of argininosuccinate synthetase 1 enzyme, which is involved in arginine synthesis. Intracellular delivery of ADI into tumor cells is suggested as an efficient approach to overcome the aforesaid drawback.

Objective: In this study, in-silico tools were used for evaluating the experimentally validated CPPs to select the best CPP candidates for the intracellular delivery of ADI.

Results: In this regard, 150 CPPs of protein cargo available at CPPsite were retrieved and evaluated by the CellPPD server. The best CPP candidates for the intracellular delivery of ADI were selected based on stability and antigenicity of the ADI-CPP fusion form. The conjugated forms of ADI with each of the three CPPs including EGFP-hcT (9-32), EGFP-ppTG20, and F(SG)4TP10 were stable and nonantigenic; thus, these sequences were introduced as the best CPP candidates for the intracellular delivery of ADI. In addition, the proposed CPPs had appropriate positive charge and lengths for an efficient cellular uptake.

Conclusion: These three introduced CPPs not only are appropriate for the intracellular delivery of ADI, but also can overcome the limitation of its therapeutic application, including short half-life and antigenicity.

背景：细胞穿透肽（CPPs）是一种短肽家族，广泛用作药物和不同治疗剂的输送载体。由于存在有价值的数据库，对经过实验验证的CPP进行计算筛选可以帮助研究人员选择更有效的CPP用于细胞间递送治疗性蛋白。人型支原体的精氨酸脱亚氨酶是一种精氨酸降解酶，目前正处于治疗几种精氨酸营养缺陷型癌症的临床试验中。然而，一些肿瘤细胞已发展出对ADI治疗的抗性。ADI抗性源自精氨酸琥珀酸合成酶1酶的过表达，该酶参与精氨酸的合成。

目的：在这项研究中，使用计算机模拟工具评估经过实验验证的CPP，以选择最佳的CPP候选物进行ADI的细胞内递送。

结果：在这方面，CellPPD服务器检索并评估了可在CPPsite获得的150 CPP蛋白质货物。基于ADI-CPP融合形式的稳定性和抗原性，选择用于ADI细胞内递送的最佳CPP候选物。ADI与包括EGFP-hcT（9-32），EGFP-ppTG20和F（SG）4TP10的三个CPP的结合形式均稳定且无抗原性。因此，这些序列被引入作为ADI细胞内递送的最佳CPP候选物。此外，建议的CPP具有适当的正电荷和适当的长度以有效吸收细胞。

结论：这三种引入的CPP不仅适合ADI的细胞内递送，而且可以克服其治疗应用的局限性，包括半衰期短和抗原性强。