Pleckstrin Homology Domain of Akt Kinase: A Proof of Principle for Highly Specific and Effective Non-Enzymatic Anti-Cancer Target

While pharmacological inhibition of Akt kinase has been regarded as a promising anti-cancer strategy, most of the Akt inhibitors that have been developed are enzymatic inhibitors that target the kinase active site of Akt. Another key cellular regulatory event for Akt activation is the translocation of Akt kinase to the cell membrane from the cytoplasm, which is accomplished through the pleckstrin homology (PH) domain of Akt. However, compounds specifically interacting with the PH domain of Akt to inhibit Akt activation are currently limited. Here we identified a compound, lancemaside A (LAN-A), which specifically binds to the PH domain of Akt kinase. First, our mass spectra analysis of cellular Akt kinase isolated from cells treated with LAN-A revealed that LAN-A specifically binds to the PH domain of cellular Akt kinase. Second, we observed that LAN-A inhibits the translocation of Akt kinase to the membrane and thus Akt activation, as examined by the phosphorylation of various downstream targets of Akt such as GSK3β, mTOR and BAD. Third, in a co-cultured cell model containing human lung epithelial cancer cells (A549) and normal human primary lung fibroblasts, LAN-A specifically restricts the growth of the A549 cells. LAN-A also displayed anti-proliferative effects on various human cancer cell lines. Finally, in the A549-luciferase mouse transplant model, LAN-A effectively inhibited A549 cell growth with little evident cytotoxicity. Indeed, the therapeutic index of LAN-A in this mouse model was >250, supporting that LAN-A is a potential lead compound for PH domain targeting as a safe anti-cancer Akt inhibitor.

尽管Akt激酶（Akt kinase）的药理学抑制已被视作极具潜力的抗癌策略，但目前已开发的绝大多数Akt抑制剂均为靶向Akt激酶活性位点的酶抑制剂。Akt激活的另一关键细胞调控事件，是Akt激酶从细胞质向细胞膜的转位，这一过程通过Akt的普列克底物蛋白同源（pleckstrin homology, PH）结构域完成。然而，当前能够特异性结合Akt的PH结构域以抑制Akt激活的化合物仍十分匮乏。本研究鉴定出一种化合物兰斯马苷A（lancemaside A，LAN-A），其可特异性结合Akt激酶的PH结构域。首先，我们对经LAN-A处理的细胞中分离得到的细胞Akt激酶进行质谱分析，结果显示LAN-A可特异性结合细胞Akt激酶的PH结构域。其次，通过检测Akt的多种下游靶标（如GSK3β、mTOR及BAD）的磷酸化水平，我们观察到LAN-A能够抑制Akt激酶向细胞膜的转位，进而阻断Akt的激活。第三，在包含人肺上皮癌细胞（A549）与正常人原代肺成纤维细胞的共培养细胞模型中，LAN-A可特异性抑制A549细胞的增殖。此外，LAN-A对多种人类癌细胞系均展现出抗增殖活性。最后，在A549-荧光素酶（luciferase）小鼠移植瘤模型中，LAN-A可有效抑制A549细胞的生长，且未观察到明显的细胞毒性。该小鼠模型中LAN-A的治疗指数大于250，证实其作为靶向PH结构域的安全抗癌Akt抑制剂，具备成为先导化合物的潜力。