Lung cancer ranks the top of malignancies that cause cancer-related deaths worldwide. The leaves of Morus alba L are traditional Chinese medicine widely applied in respiratory diseases. Our previous work has demonstrated the anti-lung cancer effect of secondary metabolites of mulberry leaf. But their mechanism of action has still not fully elucidated. We synthesized Moracin N (MAN) probe conjugated with alkyne to label lung cancer cells and identified protein targets by chemical proteomic analysis. MAN and its probe exerted similar growth-inhibitory effect on human lung cancer cells. Chemical proteomic results showed that MAN targeted the programmed death ligand 1 (PD-L1) checkpoint pathway and T cell receptor (TCR) signaling pathway, indicating its immune-regulatory function. Cell-free surface plasmon resonance (SPR) results showed the direct interaction of MAN with PD-L1 protein. Molecular docking analysis demonstrated that MAN bound to E158 residue of PD-L1 protein. MAN downregulated the expression levels of PD-L1 in a time- and dose-dependent manner and disrupted the PD-L1/programmed death 1 (PD-1) binding, including other secondary metabolites of mulberry leaves Guangsangon E (GSE) and Chalcomoracin (CMR). Human peripheral blood mononuclear cells (PBMCs) co-cultured with MAN-treated A549 cells, resulting in the increase of cluster of differentiation (CD)8⁺ Granzyme B (GZMB)⁺ T cells and the decrease of CD8⁺PD-1⁺ T cells. It suggested that MAN exerts anti-cancer effect through blocking the PD-L1/PD-1 signaling. In vivo, MAN combined with anti-PD-1 antibody significantly inhibited lung cancer development and metastasis, indicating their synergistic effect. Taken together, secondary metabolites of mulberry leaves target the PD-L1/PD-1 signaling, enhance T cell-mediated immunity and inhibit the tumorigenesis of lung cancer. Their modulatory effect on tumor microenvironment makes them able to enhance the therapeutic efficacy of immune checkpoint inhibitors in lung cancer.

肺癌位居全球导致癌症相关死亡的恶性肿瘤的首位。桑叶是广泛应用于呼吸系统疾病的中药。我们前期的工作已经证明了桑叶次生代谢产物的抗肺癌作用。但它们的作用机制尚未完全阐明。我们合成了与炔烃缀合的 Moracin N (MAN) 探针来标记肺癌细胞，并通过化学蛋白质组分析鉴定了蛋白质靶点。MAN及其探针对人肺癌细胞具有类似的生长抑制作用。化学蛋白质组学结果表明，MAN靶向程序性死亡配体1（PD-L1）检查点通路和T细胞受体（TCR）信号通路，表明其具有免疫调节功能。无细胞表面等离子共振 (SPR) 结果显示 MAN 与 PD-L1 蛋白直接相互作用。分子对接分析表明MAN与PD-L1蛋白的E158残基结合。MAN 以时间和剂量依赖性方式下调 PD-L1 的表达水平，并破坏 PD-L1/程序性死亡 1 (PD-1) 结合，包括桑叶广三贡 E (GSE) 和 Chalcomoracin 的其他次级代谢产物。共模磁共振）。人外周血单核细胞 (PBMC) 与 MAN 处理的 A549 细胞共培养，导致分化簇 (CD)8 ⁺颗粒酶 B (GZMB) ^{+ T 细胞增加，CD8 +} PD-1 ⁺ T细胞减少细胞。提示MAN通过阻断PD-L1/PD-1信号传导发挥抗癌作用。在体内，MAN与抗PD-1抗体联合显着抑制肺癌的发展和转移，表明它们具有协同作用。综上所述，桑叶的次生代谢物靶向PD-L1/PD-1信号传导，增强T细胞介导的免疫并抑制肺癌的肿瘤发生。它们对肿瘤微环境的调节作用使它们能够增强免疫检查点抑制剂对肺癌的治疗效果。