cNMP-Fc嵌合肽拮抗cN-Ⅱ逆转ALL细胞核苷类似物耐药的作用及其机制

Acute lymphoblastic leukemia (ALL) is the leading cause of death in children with malignant tumors. Nucleoside analogues are important chemotherapeutic drugs for the treatment of ALL, and the high expression of nt5c2 and its encoded cN-II protein is an important cause of drug resistance and patient relapses. Our research group have previously screened a set of cN-II-binding mimetic peptides (cNMPs) based on ribosome display technology, and after bioinformatics studies and in vitro experiments, we confirmed that a peptidomimetic could inhibit cN-II expression and reversal leukemia cell resistance, but further experiments showed that the affinity of the mimetic peptide needs to be further improved and due to the small molecular weight, it is rapidly cleared in the rats after intravenous administration. Therefore, based on the previous research, this project aims to screen and construct cNMP-Fc chimeric peptide with high affinity and purity by eukaryotic expression, and through ribosome display technology combined with error-prone PCR, to optimize its affinity and half-life problems; and study the effect and mechanism of it to cN-II expression and reversal of nucleoside analogue resistance in ALL cells; to explore the therapeutic significance of the new antagonistic strategy of cN-II as a target molecule to inhibit the efflux of nucleoside analog active metabolites in patients with resistance and relapse ALL, and lay the foundation for obtaining cNMP-Fc chimeric peptides with independent intellectual property rights and further in vivo studies of it.

急性淋巴细胞白血病(ALL)为儿童恶性肿瘤死亡的主要原因。核苷类似物是治疗ALL的重要化疗药，而nt5c2及其编码的cN-Ⅱ蛋白高表达是白血病患者核苷类似物耐药和复发的主要原因。课题组前期基于核糖体展示技术筛选出一组可与cN-Ⅱ结合的模拟肽(cNMP)，并经生物信息学和体外试验优选了一条模拟肽，其可抑制白血病细胞cN-Ⅱ表达和逆转核苷类似物耐药，但其亲和力需进一步提高，且其存在分子量小，半衰期极短的问题。因此，本项目拟在前期基础上，通过核糖体展示技术结合易错PCR，筛选构建并通过真核表达获得高亲和力和纯度的cNMP-Fc嵌合肽，以优化其亲和力和半衰期问题；并研究其通过抑制cN-Ⅱ逆转ALL细胞核苷类似物耐药的效应和机制；探讨以cN-Ⅱ为靶分子抑制核苷类似物活性代谢产物外排的新拮抗策略在ALL耐药和复发患者的治疗学意义，为获得具有自主知识产权的cNMP-Fc嵌合肽和进入临床前研究奠定基础。

急性淋巴细胞白血病(ALL)为儿童恶性肿瘤死亡的主要原因。核苷类似物是治疗ALL的重要化疗药，而nt5c2及其编码的cN-Ⅱ蛋白高表达是白血病患者核苷类似物耐药和复发的主要原因，因此以cN-Ⅱ为靶分子的新拮抗策略在ALL耐药和复发患者的疾病治疗中具有明显价值。本研究基于前期通过核糖体展示技术、生物信息学和体外试验筛选出的可与cN-Ⅱ结合的模拟肽(cNMP)，进一步通过体外定向进化获得与cN-Ⅱ高亲和力的cNMP突变体，并通过分子克隆的方法，经蛋白G亲和层析纯化系统和镍离子金属螯合(Ni-NTA)层析柱纯化系统纯化获得cNMP-Fc嵌合肽。体外试验表明了cNMP-Fc嵌合肽可抑制cN-II的寡聚，可调节细胞中嘌呤核苷酸AMP、ADP的含量，可增强6-MP和ara-c的细胞毒性作用，且可影响WT1、p53、cyclin A2和CDK2的表达，但是对notch1、nras和kras的mRNA表达无影响，可减少ALL模型小鼠血清中ASP和AST的含量。本课题的研究结果可能为ALL耐药和复发患者的药物治疗提供新的策略和途径，为相关新药的研发和临床转化奠定基础。

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