Laxiflorin B是一种天然ent-kaurene二萜类化合物，可从中国部分地区的栓翅木羊蹄甲（Isodon eriocalyx var. laxiflora）的叶子中分离得到。虽然这种化合物对多种肿瘤细胞具有强大的细胞毒活性，但Laxiflorin B的抗肿瘤靶标和分子机制尚不清楚。在这里，我们展示了Laxiflorin B在三阴性乳腺癌（TNBC）细胞上表现出强大的抗增殖和促凋亡效应。在机械水平上，我们展示了β-管蛋白（TUBB）是Laxiflorin B的细胞靶标。通过共价结合TUBB秋水仙碱结合位点的Cys239和C354残基，Laxiflorin B在体外和体内破坏了微管的完整性和结构。细胞毒性分析还显示，Laxiflorin B D环中的α，β-不饱和酮基负责介导其共价结合和抗肿瘤活性。为了评估Laxiflorin B的治疗效果，我们合成了Laxiflorin B-ALA前药，并通过腹腔注射（10 mg/kg）给予4T1原位肿瘤小鼠模型。药物治疗具有抗肿瘤效应，且没有引起明显的体重减轻或器官功能异常。我们得出结论，Laxiflorin B是一种有前景的秋水仙碱结合位点抑制剂，未来可能在TNBC治疗中得到利用。版权所有 © 2023 Elsevier B.V. 发表。

Laxiflorin B is a natural ent-kaurane diterpenoid that can be isolated from the leaves of the Isodon eriocalyx var. laxiflora, a perennial shrub native to parts of China. While this compound has potent cytotoxic activity against various tumor cells, the anti-tumor targets and molecular mechanisms of Laxiflorin B are unclear. Here, we show that Laxiflorin B exhibits strong antiproliferative and proapoptotic effects on triple-negative breast cancer (TNBC) cells. At the mechanistic level, we show that β-tubulin (TUBB) is a cellular target of Laxiflorin B. By covalently binding the Cys239 and C354 residues of the TUBB colchicine-binding site, Laxiflorin B disturbs microtubule integrity and structure in vitro and in vivo. Cytotoxicity analyses also showed that the α, β-unsaturated carbonyl in the D ring of Laxiflorin B is responsible for mediating its covalent binding and anti-tumor activity. To assess the therapeutic effects of Laxiflorin B, we synthesized a Laxiflorin B-ALA pro-drug and delivered it by intraperitoneal injection (10 mg/kg) into a 4T1 orthotopic tumor mouse model. Drug treatment had anti-tumor effects without inducing notable weight loss or organ dysfunction. We conclude that Laxiflorin B is a promising colchicine binding site inhibitor that might be exploited in the context of TNBC treatment in the future.Copyright © 2023. Published by Elsevier B.V.