Kinase experiment:

IC50 values are measured using either a standard thin-layer chromatography (TLC) assay for lipid kinase activity or a high-throughput membrane capture assay. Kinase reactions are performed by preparing a reaction mixture containing kinase, inhibitor (2% DMSO final concentration), buffer (25 mM HEPES, pH 7.4, 10 mM MgCl2), and freshly sonicated phosphatidylinositol (100 μg/mL). Reactions are initiated by the addition of ATP containing 10 μCi of γ-32P-ATP to a final concentration 10 or 100 μM, and allowed to proceed for 20 minutes at room temperature. For TLC analysis, reactions are then terminated by the addition of 105 μL 1N HCl followed by 160 μL CHCl3:MeOH (1:1). The biphasic mixture is vortexed, briefly centrifuged, and the organic phase transferred to a new tube using a gel loading pipette tip precoated with CHCl3. This extract is spotted on TLC plates and developed for 3-4 hours in a 65:35 solution of n-propanol:1M acetic acid. The TLC plates are then dried, exposed to a phosphorimager screen, and quantitated. For each compound, kinase activity is typically measured at 10-12 inhibitor concentrations representing two-fold dilutions from the highest concentration tested (100 μM). For compounds showing significant activity, IC50 determinations are repeated two to four times, and the reported value is the average of these independent measurements[4].

Cell experiment:

For proliferation assays, MOLM14, OCI-AmL3 and MV4-11 cells are cultured during 48 h at 105 cells/mL, in triplicate, in 10% FCS, without or with 0.1 or 1 μM PI-103, and then pulsed 6 h with 1μCi (37 kBq) [3H]-thymidine. The amounts of radioactivity are determined after trichloracetic acid precipitation[2].

Animal experiment:

Mice[3]Five to six month old males of either FVB/N strain or nude BALB/c strain are injected subcutaneously with one million cells in PBS. When tumor reaches between 50 and 100 mm3, mice are treated with 10 mg/kg or 70 mg/kg of PI-103 and/or 50 mg/kg sorafenib by IP injection daily. Control mice are treated with the same volume of DMSO. Tumor size and mice weight is monitored every 2 days. When mice are sacrificed, tumors are dissected and processed[3].

PI-103 is a dual inhibitor of PI3K/Akt and mTOR with IC50 value of 0.002 μM , 0.003μM, 0.003μM, 0.015μM, 0.030μM for P110α, P110β, P110γ, P110 and mTOR, respectively [1].

PI3K/AKT/mTOR pathway is an intracellular signaling pathway and plays an important role in regulating cell cycle. It has been shown that PI3K/AKT/MTOR pathway is directly related to cellular quiescence, proliferation, cancer, and longevity. And many studies have shown that activation of the PI3K/Akt signaling pathway is correlated with poor prognosis in a variety of cancers which demonstrated that inhibition of the pathway may be regarded as a promising therapy [2, 3].

PI-103 is a selective PI3K/Akt and mTORC1 inhibitor. When tested with leukemic cell lines (MOLM14, OCI-AML3 and MV4-11) with activation of PI3K/Akt, mTORC1 and ERK/MAPK signaling pathways, PI-103 blocked cells proliferation via inhibiting PI3K/Akt and mTORC1 activity in a concentration of 1μM [4]. In HCC cell line--Huh7 cells, treated PI-103 alone or combined with sorafenib inhibited cells proliferation in a dose-dependent manner through inhibition of PI3K/Akt and mTORC1 pathways [5]. When tested with U87MG glioblastoma cells, PI-103 treatment in 30 nM/L showed significant inhibition of PI3K/Akt and mTORC1 phosphorylation [1]. In neuroblastoma cell lines SK-N-BE (2), IMR-32 with amplified MYCN, PI-103 inhibited cell growth in a time- and concentration-dependent manner via inhibiting PI3K/Akt and mTORC1 activity [3].

In SK-N-BE (2) with MYCN-mutant xenografted nude mice model, treated PI-103 intraperitoneally (10 mg/kg) markedly reduced the tumor volume index and tumor weight via inhibiting PI3K/ mTOR pathways [1].

References:
[1].  Raynaud, F.I., et al., Biological properties of potent inhibitors of class I phosphatidylinositide 3-kinases: from PI-103 through PI-540, PI-620 to the oral agent GDC-0941. Mol Cancer Ther, 2009. 8(7): p. 1725-38.
[2].  Hambright, H.G., et al., Inhibition of PI3K/AKT/mTOR axis disrupts oxidative stress-mediated survival of melanoma cells. Oncotarget, 2015.
[3].  Segerstrom, L., et al., Effects of small molecule inhibitors of PI3K/Akt/mTOR signaling on neuroblastoma growth in vitro and in vivo. Int J Cancer, 2011. 129(12): p. 2958-65.
[4].  Park, S., et al., PI-103, a dual inhibitor of Class IA phosphatidylinositide 3-kinase and mTOR, has antileukemic activity in AML. Leukemia, 2008. 22(9): p. 1698-706.
[5].  Gedaly, R., et al., PI-103 and sorafenib inhibit hepatocellular carcinoma cell proliferation by blocking Ras/Raf/MAPK and PI3K/AKT/mTOR pathways. Anticancer Res, 2010. 30(12): p. 4951-8.