Inositol phospholipids have emerged as important key players in a wide variety of cellular functions. Among the seven existing inositol phospholipids, phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P 2 ) has attracted much attention in recent years due to its important role in numerous cellular signaling events and regulations, which in turn impact several human diseases. This particular lipid is recognized in the cell by specific lipid binding domains, such as the Pleckstrin-homology (PH) domain, which is also employed as a tool to monitor this important lipid. Here, we describe the synthesis and biological characterization of a small molecule that mimics the PH domain as judged by its ability to bind specifically to only PI(4,5)P 2 and effectively compete with the PH domain in vitro and in a cellular environment.The binding constant of this small molecule PH domain mimetic (PHDM) was determined to be 17.6 ± 10.1 µM, similar in potency to the PH domain. Using NIH 3T3 mouse fibroblast cells we demonstrated that this compound is cell permeable and able to modulate PI(4,5)P 2 -dependent effects in a cellular environment such as the endocytosis of the transferrin receptor, loss of mitochondria as well as stress fiber formation. This highly PI(4,5)P 2 -specific chemical mimetic of a PH domain, is not only a powerful research tool, but might also be a lead compound in future drug developments targeting PI(4,5)P 2 -dependent diseases such as Lowe syndrome.
Screening a compound library of compound 48/80 analogues, we identified 2‐[5‐(2‐chloroethyl)‐2‐acetoxy‐benzyl]‐4‐(2‐chloroethyl)‐phenyl acetate (E1) as a novel inhibitor of the phosphoinositide 3‐kinase/Akt pathway. In order to determine the mechanism of action of E1, we analysed the effect of E1 on components of the phosphoinositide 3‐kinase/Akt/mammalian target of rapamycin (mTOR) pathway. E1 demonstrated dose‐dependent and time‐dependent repression of Akt and mTOR activity in prostate and breast cancer cell lines, PC‐3 and MCF‐7, respectively. Inhibition of Akt and mTOR activity by E1 also coincided with increased c‐Jun NH2‐terminal kinase (JNK) phosphorylation. However, the mode of action of E1 is different from that of the mTOR inhibitor rapamycin. Proliferation and cell cycle analysis revealed that E1 induced cell cycle arrest and cell death in PC‐3 and MCF‐7 cells. Moreover, pretreatment of cancer cells with the JNK inhibitor SP600125 abolished the repression of Akt and mTOR activity by E1, indicating that the inhibition of Akt and mTOR by E1 is mediated through JNK activation. Consistently, E1 repressed Akt and mTOR activity in wild‐type and p38‐null mouse embryonic fibroblasts (MEFs), but not in MEFs lacking JNK1/2, and JNK‐null MEFs were less sensitive to the antiproliferative effects of E1. We further showed that E1 can function cooperatively with suboptimal concentrations of paclitaxel to induce cell death in PC‐3 and MCF‐7 cells. Taken together, these data suggest that E1 induces cancer cell death through the JNK‐dependent repression of Akt and mTOR activity and may provide a valuable compound for further development and research.
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