靶向程序性细胞死亡配体1的多肽抑制剂筛选及抗肿瘤活性评价

Screening and evaluation of the anti-tumor activity of peptide inhibitors targeting programmed death-ligand 1

目的 采用“分子对接-分子动力学-体外药理学”研究方法筛选并优化阻断程序性细胞死亡蛋白1/程序性细胞死亡配体1(programmed cell death protein 1/programmed death-ligand 1, PD-1/PD-L1)结合,获得具有较高抗肿瘤活性的多肽小分子化合物。方法 利用本团队构建的5肽化合物数据库,以及从蛋白质数据库(protein data bank, PDB)中下载的PD-L1蛋白质三维结构数据,采用Molecular Operating Environment软件进行柔性分子对接得到多肽化合物;对其中GWVI/WSA(generalized Born volume integral/weighted surface area)自由能变化值(ΔG)排名最高的化合物分子进行分子动力学计算分析,包括配体重原子位置变动的均方根偏差(root mean square error, RMSD)以及相互作用能[为兰纳-琼斯势能(Lennard-Jones potential)与库伦势能(Coulombic energy)之和]。通过均相时间分辨荧光(homogeneous time-resolved fluorescence, HTRF)技术分析配体化合物对PD-1/PD-L1相互结合的阻断作用,建立Jurkat T淋巴细胞与黑色素瘤B16-F10细胞的共培养体系,探索化合物配体对T细胞杀伤肿瘤作用的影响以及对共培养上清液中IL-2分泌水平的影响。结果 在筛选的多肽化合物中,RGGHA与RGGHH与PD-L1的结合更为稳定,其中RGGHA与PD-L1存在多种相互作用力,与PD-1竞争性结合PD-L1的Asp122、Try123、Lys124等位点阻断PD-1/PD-L1信号转导。HTRF实验表明,RGGHA对PD-1和PD-L1结合抑制率为58.38%,RGGHH为42.73%。此外,在共培养体系中,RGGHA与RGGHH能够显著增加IL-2的分泌水平,提高T细胞对于肿瘤细胞的杀伤能力,激活肿瘤免疫微环境。结论 研究发现多肽化合物RGGHA与RGGHH可以有效阻断PD-1/PD-L1相互作用,重新激活有利于抗癌的免疫反应,可以将其作为先导化合物用于新药研发。

Objective The method of"molecular docking-molecular dynamics-in vitro pharmacology"was used to screen and optimize small molecule polypeptide compounds that can block PD-1/PD-L1 binding and have high anti-tumor activity.Methods Using the 5-peptide compound database constructed by our team and the three-dimensional structure data of the PD-L1 protein downloaded from the protein database(PDB),the peptide compounds obtained by flexible molecular docking using Molecular Operating Environment software.The molecular dynamics calculation and analysis of the compounds with the highest GWVI/WSA(generalized Born volume integral/weighted surface area)free energy change value(ΔG)were carried out,including the root mean square deviation(RMSD)of the position change of the weight atom and the interaction energy(the sum of Lennard-Jones potential and Coulombic energy).The blocking effect of ligand compounds on PD-1/PD-L1 binding was analyzed by the homogeneous time-resolved fluorescence(HTRF)technique.The co-culture system of Jurkat T lymphocytes and melanoma B16-F10 cells was established to explore the effect of compound ligands on the killing effect of T cells on tumors and the effect on the secretion level of IL-2 in the co-culture supernatant.Results The selected polypeptide compounds were analyzed by molecular dynamics,and the binding of RGGHA to RGGHH and PD-L1 was stable.There are many kinds of interactions between RGGHA and PD-L1,which can compete with PD-1 ligands to bind Asp122,Try123 and Lys124 sites of the PD-L1 protein and block PD-1/PD-L1 signal transduction.HTRF experiments showed that the binding inhibition rate of RGGHA to PD-1 and PD-L1 was 58.38%,and that of RGGHH was 42.73%.In addition,we established a coculture system of Jurkat T lymphocytes and melanoma B16-F10 cells to explore the immunostimulatory effect and mechanism of peptides.The results show that RGGHA and RGGHH can significantly increase the secretion level of IL-2,improve the killing ability of T cells against tumor cells and activate the tumor immune microenvironment.Conclusions The study found that the polypeptide compounds RGGHA and RGGHH can effectively block the PD-1/PD-L1 interaction and reactivate the anti-cancer immune response,which can be used as lead compounds for new drug development.