Elucidating signaling driven by lemur tyrosine kinase 3 (LMTK3) could help drug development. Here, we solve the crystal structure of LMTK3 kinase domain to 2.1Å resolution, determine its consensus motif and phosphoproteome, unveiling in vitro and in vivo LMTK3 substrates. Via high-throughput homogeneous time-resolved fluorescence screen coupled with biochemical, cellular, and biophysical assays, we identify a potent LMTK3 small-molecule inhibitor (C28). Functional and mechanistic studies reveal LMTK3 is a heat shock protein 90 (HSP90) client protein, requiring HSP90 for folding and stability, while C28 promotes proteasome-mediated degradation of LMTK3. Pharmacologic inhibition of LMTK3 decreases proliferation of cancer cell lines in the NCI-60 panel, with a concomitant increase in apoptosis in breast cancer cells, recapitulating effects of LMTK3 gene silencing. Furthermore, LMTK3 inhibition reduces growth of xenograft and transgenic breast cancer mouse models without displaying systemic toxicity at effective doses. Our data reinforce LMTK3 as a druggable target for cancer therapy.

阐明由狐猴酪氨酸激酶 3 (LMTK3) 驱动的信号传导有助于药物开发。在这里，我们将 LMTK3 激酶结构域的晶体结构解析为 2.1Å 分辨率，确定其共有基序和磷酸化蛋白质组，揭示体外和体内 LMTK3 底物。通过高通量均相时间分辨荧光筛选与生化、细胞和生物物理分析相结合，我们确定了一种有效的 LMTK3 小分子抑制剂 (C28)。功能和机制研究表明 LMTK3 是一种热休克蛋白 90 (HSP90) 客户蛋白，需要 HSP90 才能折叠和稳定，而 C28 促进蛋白酶体介导的 LMTK3 降解。LMTK3 的药理学抑制降低了 NCI-60 组中癌细胞系的增殖，同时增加了乳腺癌细胞的凋亡，LMTK3基因沉默的重演效应。此外，抑制 LMTK3 可减少异种移植物和转基因乳腺癌小鼠模型的生长，而在有效剂量下不会表现出全身毒性。我们的数据强化了 LMTK3 作为癌症治疗的药物靶点。