多肽IMB-P1的理性设计及活性评价

Rational design and activity evaluation of the peptide IMB-P1

目的通过对多肽IMB-P1进行截短设计并进行体内和体外抗肿瘤活性评价,确定多肽IMB-P1的最小功能片段。方法分别从N端和C端对多肽IMB-P1进行截短,经固相合成法制备目标多肽。测定这些多肽对PD-1/PD-L1结合的抑制活性,在荷B16黑色素瘤小鼠模型中评价它们的体内抗肿瘤活性,并测定活性好的多肽在血浆中的稳定性及其二级结构特征。结果设计合成了6条IMB-P1截短多肽IMB-P1-1~6。经PD-1/PD-L1结合的抑制活性评价,发现IMB-P1-2、IMB-P1-3和IMB-P1-6抑制活性较好,其中多肽IMB-P1-6的抑制率最高,可达90%。在小鼠体内抗肿瘤活性研究发现IMB-P1-2的抑瘤作用与IMB-P1相当,其他多肽的抑瘤活性都有所下降。经过HPLC检测发现多肽IMB-P1-2在血浆中的稳定性优于IMB-P1-3和IMB-P1-6。多肽IMB-P1无论是在水中还是在SDS环境中均呈无规则卷曲状态,而多肽IMB-P1-2在水中形成了α-螺旋结构。结论鉴定出与多肽IMB-P1体内抗肿瘤活性相当的更短多肽IMB-P1-2及其二级结构特征,为将来进一步提高多肽活性的修饰策略提供了锁定其α-螺旋构象的方向。

Objective To determine the minimal functional fragment of peptide IMB-P1 by evaluating antitumor activity of the truncated peptides of IMB-P1 in vitro and in vivo.Methods The peptide IMB-P1 was truncated from the N-terminal and C-terminal respectively,and six target peptides(IMB-P1-1~6)were prepared by solid-phase synthesis.The antitumor activities of these shortened peptides were evaluated by measuring the inhibitory activity of PD-1/PD-L1 binding in vitro and the effects on suppressing tumor growth in mouse model of B16 melanoma.The stability in plasma and secondary structure of the peptides with better activity were determined.Results In the study,six IMB-P1 truncated peptides,IMB-P1-1~6,were designed and synthesized.IMB-P1-2,IMB-P1-3 and IMB-P1-6 showed better inhibitory activity through evaluating the binding of PD-1/PD-L1.The inhibitory rate of IMB-P1-6 was the highest,up to 90%,among the three peptides.The in vivo results showed that tumor suppression effect of IMB-P1-2 was equivalent to IMB-P1,but other peptides presented lower activity.The peptide IMB-P1-2 had a better stability in plasma than IMB-P1-3 and IMB-P1-6 by HPLC analysis.IMB-P1 formed a random coil structure both in water and SDS,while IMB-P1-2 could maintain anα-helix structure in water by the secondary structure analysis.Conclusion The truncated peptide IMB-P1-2 had remarkably similar anti-tumor activity to its parent peptide IMB-P1,and its secondary structure was determined.The study provides a direction of lockingα-helix conformation for further modification strategies to improve peptide activity in the future.