Department of Integrative Biology and Pharmacology, University of Texas Health Science Center, Houston, TX 77030, USA.
Proc Natl Acad Sci U S A. 2013 Jun 18;110(25):10201-6. doi: 10.1073/pnas.1300016110. Epub 2013 Jun 4.
Aberrant signaling by oncogenic mutant rat sarcoma (Ras) proteins occurs in ∼15% of all human tumors, yet direct inhibition of Ras by small molecules has remained elusive. Recently, several small-molecule ligands have been discovered that directly bind Ras and inhibit its function by interfering with exchange factor binding. However, it is unclear whether, or how, these ligands could lead to drugs that act against constitutively active oncogenic mutant Ras. Using a dynamics-based pocket identification scheme, ensemble docking, and innovative cell-based assays, here we show that andrographolide (AGP)--a bicyclic diterpenoid lactone isolated from Andrographis paniculata--and its benzylidene derivatives bind to transient pockets on Kirsten-Ras (K-Ras) and inhibit GDP-GTP exchange. As expected for inhibitors of exchange factor binding, AGP derivatives reduced GTP loading of wild-type K-Ras in response to acute EGF stimulation with a concomitant reduction in MAPK activation. Remarkably, however, prolonged treatment with AGP derivatives also reduced GTP loading of, and signal transmission by, oncogenic mutant K-RasG12V. In sum, the combined analysis of our computational and cell biology results show that AGP derivatives directly bind Ras, block GDP-GTP exchange, and inhibit both wild-type and oncogenic K-Ras signaling. Importantly, our findings not only show that nucleotide exchange factors are required for oncogenic Ras signaling but also demonstrate that inhibiting nucleotide exchange is a valid approach to abrogating the function of oncogenic mutant Ras.
致癌突变鼠肉瘤（Ras）蛋白的异常信号发生在所有人类肿瘤的约 15%中，但小分子直接抑制 Ras 仍然难以实现。最近，已经发现了几种小分子配体，它们通过干扰交换因子结合直接与 Ras 结合并抑制其功能。然而，尚不清楚这些配体是否可以或如何导致针对组成性激活的致癌突变 Ras 的药物。在这里，我们使用基于动力学的口袋识别方案、整体对接和创新的基于细胞的测定，表明穿心莲内酯（AGP）——一种从穿心莲中分离出的双环二萜内酯——及其苄叉衍生物结合到 Kirsten-Ras（K-Ras）上的瞬态口袋，并抑制 GDP-GTP 交换。正如预期的那样，AGP 衍生物是交换因子结合抑制剂，可减少急性 EGF 刺激下野生型 K-Ras 的 GTP 加载，并伴有 MAPK 激活减少。然而，令人惊讶的是，AGP 衍生物的长期治疗也减少了致癌突变 K-RasG12V 的 GTP 加载和信号转导。总之，我们的计算和细胞生物学结果的综合分析表明，AGP 衍生物直接与 Ras 结合，阻断 GDP-GTP 交换，并抑制野生型和致癌 K-Ras 信号。重要的是，我们的发现不仅表明核苷酸交换因子是致癌 Ras 信号所必需的，而且还证明抑制核苷酸交换是一种有效的方法来消除致癌突变 Ras 的功能。
