D-3-Deoxy-phosphatidylinositol derivatives have cytotoxic activity against various human cancer cell lines. These phosphatidylinositols have a potentially wide array of targets in the phosphatidylinositol-3-kinase (PI3K)/Akt signaling network. To explore the specificity of these types of molecules, we have synthesized D-3-deoxy-dioctanoylphosphatidylinositol (D-3-deoxydiC 8 PI), D-3,5-dideoxy-diC 8 PI and D-3-deoxy-dioctanoylphosphatidylinositol-5-phosphate and their enantiomers, characterized their aggregate formation by novel high resolution field cycling 31 P NMR, and examined their susceptibility to phospholipase C (PLC) and their effects on the catalytic activity of PI3K and PTEN against diC 8 PI and dioctanoylphosphatidylinositol-3-phosphate substrates, respectively, as well as their ability to induce the death of the U937 human leukemic monocyte lymphoma cells. Of these molecules, only D-3-deoxy-diC 8 PI was able to promote cell death; it did so with an IC 50 of 40 μM, well below the CMC of 0.4 mM. Under these conditions, there was little inhibition of PI3K or PTEN observed in assays of recombinant enzymes (although the complete series of deoxy-PI compounds did provide insights into ligand binding by PTEN). The D-3-deoxydiC 8 PI was a poor substrate and not an inhibitor of the PLC enzymes. The in vivo results are consistent with the current thought that the PI analogue acts on Akt1 since the transcription initiation factor eIF4e, which is a downstream signaling target of the PI3K/Akt pathway, exhibited reduced phosphorylation on Ser209. Phosphorylation of Akt1 on Ser473, but not Thr308, was reduced. Since the potent cytotoxicity for U937 cells is completely lost with the L-3-deoxy-diC 8 PI as well as with modification of the hydroxyl group at the inositol C5 (either replacing the -OH with a hydrogen or phosphorylating it) in D-3-deoxy-diC 8 PI, both chirality of the phosphoinositol moiety and the hydroxyl group at C5 are major determinants of 3-deoxy-PI binding to its target in cells.

one revolution, the b returns to the ATP waiting state again; however, it has not been identified where the bound Pi is released, although it was suggested to occur at þ200 or þ320 from the ATP-binding angle. In this study, we observed the rotations of the hybrid F 1 -ATPase, a 3 b(WT) 2 b(E190D) 1 g with the highspeed camera. At þ320 from the ATP binding angle of the incorporated mutant b(E190D), the clear pause of~7msec was observed as reported previously (Ariga et al. Nat. Struct. Mol. Biol.). When high concentrations of Pi was added to the solution, the time constant of the new reaction was specifically prolonged upon addition of Pi, suggesting that Pi was released at þ320 . Other lines of experiments also support this result.

D-3-deoxy-phosphatidylinositol derivatives have a potentially wide array of targets in the phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway. As such, these phosphatidylinositol molecules have been shown to have cytotoxic activity against a variety of human cancer cell lines. Here, we demonstrate that the D-3-deoxy-dioctanoyl-PI (D-diC8PI), but not the L-isomer or D-3,5-dideoxy-diC8PI, is able to induce the death of the U-937 human leukemic monocyte lymphoma cell line, even at low concentrations (<50 μM). In order to further investigate the mechanism of the cytotoxicity of D-3-deoxy-diC8PI we examined its effect on the activity of various molecules in the PI3K/Akt pathway. In vitro, low concentrations (<50 μM) of D-3-deoxy-diC8PI have no significant effect on the activity of PI3K or PTEN. However, incubation of U-937 cells with D-3-deoxy-diC8PI at these concentrations results in significant decreases in phosphorylation of key components of the PI3K/Akt signalling pathway, including the transcription initiation factor eIF4e and the translation factor S6 ribosomal protein. These data further delineate the mechanism of action of such cytotoxic D-3-deoxy-PI compounds.