基于溶酶体途径下调免疫检查点PD-L1的莲心碱类新化合物IMB-29抗非小细胞肺癌作用及机制研究

Among the most promising approaches to activating therapeutic antitumour immunity is the blockade of immune checkpoints. Programmed cell death protein-1 (PD-1) and programmed cell death ligand-1 (PD-L1) are key targets in the treatment of cancer, but current antibody-based drugs against this pathway have inherent drawbacks that may limit their effectiveness. Therefore, non-antibody, small moleculars cancer immunotherapies against PD-1/PD-L1 pathway is urgently demanded..During our screening program for natural anticancer drugs down-regulating PD-L1, we have recently discovered that IMB-29, a novel liensinine derivative, has potent anti- non-small cell lung cancer (NSCLC) activity. Our primary results demonstrated that IMB-29 significantly increased the cytotoxity of co-cultured T cells and NKcells by degradation of PD-L1 expressed on the NSLC cells. Meanwhile, we also found that IMB-29 regulated the lysosomal biogenesis in NSCLC cell by upregulation of the transcription activity of TFEB as well as controlling the expression of PKCδ. Therefore, we hypothesize that IMB-29 regulates lysosomal biogenesis through PKCδ-TFEB pathways. This proposal is novel, as it will demonstrate the effect of IMB-29-induced lysosomal-dependent PD-L1 degradation, delineate the molecular mechanism by which IMB-08B regulates TFEB activity through PKCδ-TFEB signaling pathway, investigate the target protein of IMB-29, evaluate the antitumor effect of IMB-29 in vivo, preliminary pharmacokinetics and safety of IMB-29 with the use of multiple cellular, molecular biotechnologies and animal models.This study will clarify a new role of IMB-29 in cancer immunotherapies against PD-1/PD-L1 pathway, and provide a theoretical basis for the mechanism study of liensinine derivatives against NSCLC.

免疫检查点阻断剂已成为肿瘤免疫治疗的热点，开发靶向下调PD-L1的小分子药物是肺癌等恶性肿瘤免疫治疗的新方向。前期研究发现，我们自主创新、具有全新骨架结构的莲心碱类化合物IMB-29能显著下调肺癌细胞中PD-L1的表达，促进共培养的T/NK细胞对NSCLC细胞的杀伤作用，在小鼠体内也显示出较好的抗肿瘤活性，并具有较好的安全性。本项目拟在此原创性工作基础上，以PD-L1可发生溶酶体途径降解的新发现以及IMB-29调控PKCδ表达为依据，深入研究：（1）IMB-29通过溶酶体途径降解PD-L1；（2）IMB-29通过PKCδ-TFEB途径调控溶酶体生物合成的机制；（3）鉴定IMB-29的直接靶蛋白；（4）移植瘤及自发性小鼠肺癌模型评价IMB-29的体内抗肿瘤效果；（5）评价IMB-29的药代和安全性，为将IMB-29研发成靶向PD-L1的新型小分子肿瘤免疫治疗候选物奠定坚实的基础。

免疫检查点阻断剂已成为肿瘤免疫治疗的热点，开发靶向下调PD-L1的小分子药物是肺癌等恶性肿瘤免疫治疗的新方向。天然产物是创新抗肿瘤药物的主要来源之一，从中寻找能降低PD-L1水平发挥抗肿瘤作用的小分子药物具有广阔的应用前景。本研究从多个角度阐明了天然活性产物（如莲心碱类化合物IMB-29、小檗碱、土贝母苷甲、酯蟾毒配基RB等）的抗肿瘤作用及分子机制。如阐明了酯蟾毒配基RB通过激活PKC抑制GSK3的活化，抑制NF-kB通路诱发胰腺癌细胞凋亡的机理，为RB在胰腺癌治疗中的潜在应用提供了依据；揭示了莲心碱衍生物IMB-29 通过PKC-ZKSCAN3途径激活溶酶体生物合成降解PD-L1的机制；解析了苦豆碱衍生物SA-49通过调控PKC-GSK3通路促进MITF激活，诱导PD-L1发生溶酶体依赖性降解，从而发挥抗肺癌作用的机制；同时，我们发现小檗碱通过直接作用于去泛素化酶CSN5并抑制其活性，促进PD-L1发生泛素-蛋白酶体途径降解，进而重塑肿瘤免疫微环境发挥肿瘤免疫治疗作用的机制；此外，我们还发现土贝母苷甲通过促进PD-L1发生TFEB依赖性溶酶体途径降解，从而增强CD8+T细胞对肿瘤细胞的杀伤活性发挥抗肿瘤免疫作用。以上研究为将这些天然活性化合物发展成为新型抗肿瘤免疫药物提供了重要的科学依据，促进了我国天然产物创新药物研究。