基于裂解肽ABH3发展靶向治疗耐药性骨肉瘤的多肽药物研究

Osteosarcoma (OS) is the most common primary malignancy of bone, afflicting predominantly children and young adults. Despite significant strides in chemotherapy, drug resistance of osteosarcoma is one of the leading cause of death. Lytic peptides emerge as a new class of promising anti-resistance reagents due to their membrane disruption mechanism. Based on this, we developed a lytic peptide, ABH3, which was engineered from the original BH3 sequence. ABH3 exhibited wide-spectrum growth inhibition over various cancer cell lines in vitro and in HeLa human cervical tumor xenografts in vivo, in which ABH3 showed the highest toxicity towards osteosarcoma cell line U-2OS. However, the stability of ABH3 was bad when treated with trypsin and it had non-specific toxicity to normal cell lines. The terminal aspartic method we developed before could improve the stability, helicity, cell-membrane permeability and activity of the peptides. And phage display can support the targeted therapy by screening osteosarcoma cell specific binding peptides. So, we can realize the goal of targeted therapy of drug-resistance osteosarcoma as follows. First, we will stabilize ABH3 through terminal aspartic method. Second, we will develop a specific-binding peptide of osteosarcoma by phage display. Last, we will combine the two parts above and test its activity with drug-resistance osteosarcoma cells both in vitro and in vivo. The success of this program will introduce a new strategy for the targeted therapy of drug-resistance osteosarcoma.

骨肉瘤是一种青少年高发的恶性肿瘤，耐药性的产生是其化疗失败的重要原因。裂解肽通过直接作用于细胞膜杀死癌细胞，有望克服耐药性的难题。本项目在前期研究中发展了基于BH3-only蛋白的裂解肽ABH3，对多种肿瘤细胞均有较强的杀伤作用，其中骨肉瘤细胞U-2OS效果最佳。但ABH3的稳定性差，且对非肿瘤细胞也有毒性。多肽成环可以提高稳定性，本项目前期发展了末端天冬氨酸法，该方法不仅可以提高多肽的稳定性，还能有效提高其活性；而通过噬菌体筛选技术，获得骨肉瘤特异性结合肽（OSBP），可以实现靶向治疗。基于此，本项目首先将通过末端天冬氨酸法环化ABH3获得TD-BH3，提高多肽的稳定性和活性；再通过成熟的噬菌体筛选技术，获得OSBP；最后，合成TD-BH3-OSBP，研究其对耐药性骨肉瘤的靶向治疗效果，并通过耐药性骨肉瘤动物模型对治疗效果进行验证。本项目有望发展一种治疗耐药性骨肉瘤的新方法。

骨肉瘤是一种青少年高发的恶性肿瘤，耐药性的产生是其化疗失败的重要原因。裂解肽通过直接作用于细胞膜杀死癌细胞，有望克服耐药性的难题。本项目在前期研究中发展了基于BH3-only蛋白的裂解肽ABH3，对多种肿瘤细胞均有较强的杀伤作用，其中骨肉瘤细胞U-2OS效果最佳。但ABH3的稳定性差，多肽成环可以提高稳定性，本项目前期发展了末端天冬氨酸法，该方法不仅可以提高多肽的稳定性，还能有效提高其活性，因此项目初期通过末端天冬氨酸法合成了TD-BH3，并测定了其体内体外的活性，结果显示TD-BH3较ABH3并未有显著的活性提高。因此继续合成了一系列由KLA单元构成的裂解肽，并通过细胞实验对其活性进行了筛选，其中Uk14活性最佳。由于裂解肽通过破裂细胞膜杀死癌细胞，因此其亦能杀死正常细胞，为了将其应用于肿瘤的治疗，必须对其进行靶向优化。本项目首先筛选了一系列的骨肉瘤亲和多肽，但在实际实验过程中，这些多肽均未表现出良好的骨肉瘤细胞亲和能力。由于骨肉瘤细胞较正常细胞高表达MMP-2，因此本项目设计将裂解肽与带负电的氨基酸通过MMP-2特异水解底物多肽偶联，合成了e8-Uk14。基于此设计，在正常组织细胞中，由于MMP-2含量低，带负电的天冬氨酸可以中和裂解肽的正电性，降低其毒性；而在癌组织中，MMP-2高表达，可以从e8-Uk14中释放出自由的裂解肽，将癌细胞杀死。通过体外实验，我们证明了e8-Uk14对正常细胞的低毒性，动物实验证明了e8-Uk14的抗癌活性。本项目可有望发展一种治疗耐药性骨肉瘤的新方法。

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