Although macroautophagy/autophagy is involved in hepatocellular carcinoma (HCC) initiation and development and has been identified as a mechanism of HCC therapy resistance, the role of ULK1 (unc-51 like autophagy activating kinase 1) in HCC remains unclear. Here, we report that both knockdown and knockout of ULK1 inhibited human HCC cell proliferation and invasion, and Ulk1 deletion abrogated tumor growth in a xenograft mouse model. Furthermore, ULK1 ablation in combination with sorafenib significantly inhibited HCC progression compared with sorafenib treatment alone or vehicle control. To identify candidate ULK1 inhibitors, we used a structure-based virtual docking approach to screen 3428 compounds. Among these compounds, XST-14 showed the highest affinity for the ULK1 protein and specifically blocked ULK1 kinase activity. Moreover, the Lys46, Tyr94 and Asp165 amino acid residues of ULK1 were required for its binding to XST-14 according to molecular docking and mutagenesis experiments. Functional assays revealed that XST-14 blocked autophagy and subsequently induced apoptosis and inhibited growth in HCC cells. More importantly, XST-14 acted synergistically with sorafenib to attenuate HCC progression by inhibiting sorafenib-induced autophagy activation both in vitro and in vivo. In addition, XST-14 was well tolerated and exhibited favorable drug metabolism and pharmacokinetic properties and low toxicity in mice. In summary, our study determined that ULK1 may represent a new therapeutic target for HCC and that targeting ULK1 in combination with sorafenib treatment may serve as a promising interventional strategy for treating HCC.

Abbreviations: 3MA: 3-methyladenine; ADV: AutoDock Vina; ATP: adenosine triphosphate; EdU: 5-ethynyl-2′-deoxyuridine; ESI: electrospray ionization; HCC: hepatocellular carcinoma; IC₅₀: half maximal inhibitory concentration; KD: kinase domain; q.o.d., every other day; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SPR, surface plasmon resonance.

尽管巨自噬/自噬参与肝细胞癌 (HCC) 的发生和发展，并且已被确定为 HCC 治疗抵抗的机制，但 ULK1（unc-51 类似自噬激活激酶 1）在 HCC 中的作用仍不清楚。在这里，我们报告敲除和敲除ULK1 都抑制了人类 HCC 细胞的增殖和侵袭，并且Ulk1缺失消除了异种移植小鼠模型中的肿瘤生长。此外，ULK1与索拉非尼单独治疗或载体对照相比，消融联合索拉非尼可显着抑制 HCC 进展。为了确定候选 ULK1 抑制剂，我们使用基于结构的虚拟对接方法来筛选 3428 种化合物。在这些化合物中，XST-14 对 ULK1 蛋白表现出最高的亲和力，并特异性阻断 ULK1 激酶活性。此外，根据分子对接和诱变实验，ULK1 的 Lys46、Tyr94 和 Asp165 氨基酸残基是其与 XST-14 结合所必需的。功能分析表明，XST-14 阻断自噬，随后诱导细胞凋亡并抑制 HCC 细胞的生长。更重要的是，XST-14 与索拉非尼协同作用，通过在体外抑制索拉非尼诱导的自噬激活来减缓 HCC 进展。和体内。此外，XST-14 具有良好的耐受性，在小鼠中表现出良好的药物代谢和药代动力学特性以及低毒性。总之，我们的研究确定 ULK1 可能代表 HCC 的新治疗靶点，并且靶向 ULK1 联合索拉非尼治疗可能作为治疗 HCC 的有希望的介入策略。

缩写： 3MA：3-甲基腺嘌呤；ADV：AutoDock Vina；ATP：三磷酸腺苷；EdU：5-乙炔基-2'-脱氧尿苷；ESI：电喷雾电离；HCC：肝细胞癌；IC ₅₀：半数最大抑制浓度；KD：激酶结构域；qod，每隔一天；SDS-PAGE，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳；SPR，表面等离子体共振。