Nitidine chloride inhibits mTORC1 signaling through ATF4-mediated Sestrin2 induction and targets IGF2R for autophagic degradation

Nitidine chloride (NC), a natural phytochemical alkaloid derived from Zanthoxylum nitidum (Roxb.) DC, exhibits multiple bioactivities in cancer, infection disease, and other therapeutic areas. However, the primary targets of NC and the mechanism of action (MOA) have not been defined explicitly. Here, we report that NC inhibit mTORC1 activity by targeting amino acid sensing signaling through activating transcription factor 4 (ATF4)-mediated Sestrin2 induction, and it efficiently induces autophagy in cells and tumor xenograft. NC displays potent cytotoxicity against various cancer cells and inhibits xenograft tumor growth of B16 melanoma. Furthermore, we identified insulin-like growth factor 2 receptor (IGF2R) as a direct target of NC by drug affinity responsive target stability (DARTS) technique. NC may functional as a IGF2R antagonist to inhibit growth factor-mediated mTORC1 activation. This study suggested that NC targets mTORC1 signaling through nutrients sensing and growth factor receptor binding, providing mechanistic insights into the MOA of NC. HEK-293T cells were treated with DMSO and 10μM nitidine chloride for 12 hours, with 3 replicates set for each treatment. RNA was extracted and subjected to high-throughput sequencing.

氯化两面针碱（Nitidine chloride, NC）是一种源自两面针[Zanthoxylum nitidum (Roxb.) DC]的天然植物生物碱，在癌症、感染性疾病及其他治疗领域展现出多种生物活性。然而，NC的主要作用靶点及作用机制（MOA）尚未被明确阐明。本研究发现，NC通过靶向氨基酸感知信号通路，经由激活转录因子4（ATF4）介导的Sestrin2诱导，抑制哺乳动物雷帕霉素靶蛋白复合物1（mTORC1）的活性，并可在细胞及肿瘤异种移植模型中有效诱导自噬。NC对多种癌细胞均表现出强效细胞毒性，且可抑制B16黑色素瘤的异种移植瘤生长。此外，本研究通过药物亲和响应靶点稳定性（DARTS）技术，确认胰岛素样生长因子2受体（IGF2R）为NC的直接作用靶点。NC可作为一种IGF2R拮抗剂，抑制生长因子介导的mTORC1激活。本研究表明，NC通过营养感知及生长因子受体结合靶向mTORC1信号通路，为阐明其作用机制提供了新的理论依据。本研究设置每组3次生物学重复，以二甲基亚砜（DMSO）及10μM氯化两面针碱处理HEK-293T细胞12小时，随后提取总RNA并进行高通量测序。