PI-103 is a potent, cell-permeable, ATP-competitive inhibitor of PI3K family members (IC50s: 2/3/3/15/30/23 nM for p110α/β/δ/γ, mTOR, and DNA-PK).

p110δ:3 nM (cell free), p110β:3 nM (cell free), p110α:2 nM (cell free), mTOR:30 nM (cell free), p110γ:15 nM (cell free), DNA-PK:23 nM (cell free)

PI-103 potently inhibited p110α (IC50: 15 nmol/L. PI-103 exhibited potent growth inhibition in each of the cell lines examined, with activity in the submicromolar range [1]. PI-103 induced proliferative arrest in a panel of glioma cell lines assayed by flow cytometry. PI-103 uniquely and potently inhibits both complexes of mTOR: the rapamycin-sensitive mTORC1 (IC50: 0.02 μM) and the rapamycin-insensitive mTORC2 (IC50: 0.083 μM). PI-103 (IC50 < 0.1 μM) was blocking the phosphorylation of p70 S6 kinase, ribosomal protein S6, and 4E-BP1, downstream markers of mTOR signaling [2].

When tumors reached 50–100 mm^3, animals were randomized and treated with vehicle or PI-103. PI-103 showed significant activity in vivo, reducing average tumor size by 4-fold after 18 days. Preclinical treatment of glioma xenografts with PI-103 blocked proliferation without inducing apoptosis [2]. PI-103 (10 mg/kg) treatment promoted a significant in vivo tumor growth compared with the DMSO treated mice. PI-103 (70 mg/kg) also promoted a significant in vivo tumor growth [3].

Phosphatidylinositide 3-kinase inhibitory activity was determined using a scintillation proximity assay in the presence of 1 μmol/L ATP. Inhibition of mTOR protein kinase was determined using a TR-FRET-based LanthaScreen method. Compounds were assayed at a maximum concentration of 10 μmol/L in the presence of 1 μmol/L ATP, and IC50 values were determined using GraphPad Prism software [1].

Human glioma cell lines were obtained from the Brain Tumor Research Center at UCSF. Cells were harvested and fixed, treated with RNAase and propidium iodide, and filtered through 95 mM nylon mesh. Ten thousand stained nuclei were analyzed in a FACS Calibur flow cytometer. DNA histograms were modeled offline using Modifit-LT software. For crystal violet staining, 10^5 cells were seeded in 12-well plates in the presence or absence of PI-103 [2].

Five to six-month-old males of either FVB/N strain or nude BALB/c strain were injected subcutaneously with one million cells in PBS. When the tumor reached between 50 and 100 mm^3, mice were treated with the inhibitors. Treatments were done by IP injection daily with 10 mg/kg or 70 mg/kg of PI-103 and/or 50 mg/kg sorafenib. Control mice were treated with the same volume of DMSO. Tumor size and mice weight was monitored every 2 days. Tumor volume was calculated with the equation (d^2*D) (p/6). When mice were sacrificed, tumors were dissected and processed. For immunosuppression experiments, mice were treated with rapamycin (1 mg/kg) or LY294002 (25 mg/kg) by a daily IP injection for a total of 8 days [3].