Zhang Jing, Sun Yue, Zhong Li-Ye, Yu Nan-Nan, Ouyang Lan, Fang Run-Dong, Wang Yang, He Qing-Yu
The First Affiliated Hospital, Jinan University, Guangzhou 510632, China.
MOE Key Laboratory of Tumor Molecular Biology and Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, China.
Comput Struct Biotechnol J. 2020 Nov 30;18:3936-3946. doi: 10.1016/j.csbj.2020.11.033. eCollection 2020.
Rab5 is a small GTPase that plays a crucial role in oncogenic signal transduction, which was considered as an attractive target for cancer therapy. Rapid GDP/GTP exchange in the packet of Rab5 sustains its high activity for promoting cancer progression. However, Rab5 currently remains undruggable due to the lack of specific inhibitor. Herein, we reported the discovery of a novel Rab5 inhibitor, neoandrographolide (NAP), by using high-throughput virtual screening with a natural product library containing 7459 compounds, which can occupy the surface groove of Rab5, competing with GDP/GTP for the binding. Ser34 is the most important residue in the groove of Rab5, as it forms most hydrogen-bond interactions with GDP/GTP or NAP, and mutation of Ser34 decreased the stabilization of Rab5. Moreover, fluorescence titration experiment and isothermal titration calorimetry (ITC) assay revealed a direct binding between NAP and Rab5. Biochemical and cell-based assays showed that NAP treatment not only diminished the activity of Rab5, but also suppressed cell growth of cancer cell. This finding firstly identifies NAP as a novel inhibitor of Rab5, which directly binds with Rab5 by occupying the GDP/GTP binding groove to suppress its functions, highlighting a great potential of NAP to be developed as a chemotherapeutic agent in cancer therapy.
Rab5是一种小GTP酶，在致癌信号转导中起关键作用，被认为是癌症治疗的一个有吸引力的靶点。Rab5分子包中快速的GDP/GTP交换维持其促进癌症进展的高活性。然而，由于缺乏特异性抑制剂，Rab5目前仍然难以成药。在此，我们报告通过使用包含7459种化合物的天然产物库进行高通量虚拟筛选，发现了一种新型Rab5抑制剂新穿心莲内酯（NAP），它可以占据Rab5的表面凹槽，与GDP/GTP竞争结合。Ser34是Rab5凹槽中最重要的残基，因为它与GDP/GTP或NAP形成最多的氢键相互作用，Ser34的突变降低了Rab5的稳定性。此外，荧光滴定实验和等温滴定量热法（ITC）分析揭示了NAP与Rab5之间的直接结合。生化和基于细胞的分析表明，NAP处理不仅降低了Rab5的活性，还抑制了癌细胞的生长。这一发现首次确定NAP是一种新型的Rab5抑制剂，它通过占据GDP/GTP结合凹槽与Rab5直接结合以抑制其功能，突出了NAP作为癌症治疗中一种化疗药物开发的巨大潜力。
