Daam1（Dishevelled相关形态发生激活蛋白1）是一种Wnt/PCP信号蛋白，能够参与细胞骨架重组并在某些肿瘤中出现异常活化。Daam1与癌症转移密切相关，是癌症治疗的潜在靶点。然而，尚未发现自然小分子能够靶向Daam1。我们通过Sybyl分子模拟对几种可能与Daam1结合的天然小分子进行了筛选。作为小细胞肺癌治疗的一线药物，我们选择依托泊苷进行进一步的研究。然后，我们使用微缩比例热泳动光学（MST）来验证依托泊苷和Daam1之间的相互作用。我们将小细胞肺癌H446细胞和乳腺癌MCF-7细胞处理依托泊苷，并进行Western blot检测Daam1的表达。我们通过体外的CCK-8实验和体内的移植瘤小鼠模型来确定依托泊苷对细胞增殖的影响。我们使用创口愈合实验和Boyden小室实验来评估依托泊苷对肿瘤细胞迁移和侵袭能力的影响。我们通过鸟笼素免疫荧光染色实验来可视化依托泊苷对微丝装配的影响。最后，我们通过半衰期实验和溶酶体标记物LAMP1的免疫荧光染色来检测依托泊苷诱导小细胞肺癌细胞下调Daam1表达的可能机制。 我们通过Sybyl分子模拟对自然化学物质库进行筛选，发现依托泊苷可以与Daam1进行虚拟交互作用。MST验证依托泊苷直接与Daam1的FH2结构域结合。依托泊苷显著下调了小细胞肺癌H446细胞和乳腺癌MCF-7细胞中Daam1的表达。此外，270μmol/L的依托泊苷能够大幅度抑制H446细胞和MCF-7细胞的增殖、迁移和侵袭。免疫荧光染色实验显示依托泊苷诱导H446细胞和MCF-7细胞的微丝解聚，但过表达Daam1可以抵消这种作用。在移植小鼠中，尾静脉注射5、10、20mg/kg（药物/体重）的依托泊苷显著抑制了皮下肿瘤的增殖。依托泊苷促使Daam1缩短其半衰期，进入溶酶体降解通路，并最终导致Daam1表达的下调。 依托泊苷是一种新型的自然小分子，可以靶向Daam1。依托泊苷能够抑制小细胞肺癌和乳腺癌细胞的增殖、迁移和侵袭，并且在小细胞肺癌中还抑制了肿瘤的增殖。版权所有 ©2023 Elsevier Inc.

Daam1 (Dishevelled-associated activator of morphogenesis 1) is a Wnt/PCP signaling protein that engages in cytoskeleton reorganization and is abnormally activated in certain tumors. Daam1 is closely related to cancer metastasis, which is expected to become a target for cancer treatment. However, the natural small molecules targeting Daam1 have not been identified.We screened several natural small molecules that may bind to Daam1 by Sybyl molecular simulation docking technique. As a first-line drug for the treatment of small cell lung cancer, etoposide was chosen for further investigation. Next, we used Micro Scale Thermophoresis (MST) to verify the interaction of etoposide and Daam1. Small cell lung cancer H446 cells and breast cancer MCF-7 cells were treated with etoposide and subjected to Western blotting to measure the Daam1 expression. The effect of etoposide on cell proliferation was determined by CCK-8 assay in vitro and by a tumor-bearing mouse model in vivo. Wound healing assay and Boyden chamber assay were used to evaluate the role of etoposide in the migration and invasion ability of tumor cells. The effect of etoposide on the microfilament assembly was visualized by immunofluorescence staining with phalloidine. Finally, the possible mechanism of down-regulation of Daam1 expression after etoposide-induced small cell lung cancer cells was detected by a half-life experiment and immunofluorescence staining with lysosomal marker LAMP1.Sybyl molecular modeling docking technique was performed to screen a natural chemical library for molecules that bound to the FH2 domain of Daam1 and found etoposide was virtually interacted with Daam1. MST validated etoposide directly bound to the FH2 domain of Daam1. Etoposide significantly down-regulated the expression of Daam1 in small cell lung cancer H446 cells and breast cancer MCF-7 cells. Moreover, 270 μmol/L etoposide largely inhibited the proliferation, migration, and invasion of H446 cells and MCF-7 cells. Immunofluorescence staining experiments revealed that etoposide induced the disassembly of microfilaments in H446 cells and MCF-7 cells, which were rescued by Daam1 overexpression. In nude mice transplanted with H446 cells, 5, 10, 20 mg/kg etoposide (drug/weight) injected via tail vein largely retarded the proliferation of subcutaneous tumors. Etoposide induced Daam1 to shorten its half-life and enter the lysosome degradation pathway, and eventually leading to the downregulation of Daam1 expression.Etoposide is a novel natural small molecule targeting Daam1. Etoposide inhibits the proliferation, migration and invasion of small cell lung cancer cells and breast cancer cells, and also suppresses tumor proliferation of small cell lung cancer in vivo.Copyright © 2023. Published by Elsevier Inc.