Protein Kinase Assays

PI3Kα, β, and δ proteins were composed of the iSH2 domain of p85 NH2-terminally fused to the full-length protein p110 protein, with the exception of α that also did not contain the last 20 amino acids. PI3Kγ was produced as full-length protein deleted for its first 144 amino acids. All constructs were fused to a COOH-terminal His tag for convenient purification and then cloned into the pBlue-Bac4.5 (for α, β, and δ isoforms) or pVL1393 (for γ isoform) plasmids. The different vectors were then cotransfected with BaculoGold WT genomic DNA. Compounds were tested for their activity against PI3K using a Kinase-Glo assay. The kinase reaction was done in 384-well black plate (Corning). Each well was loaded with 50 nL of test items (in 90% DMSO) and 5 μL reaction buffer [10 mmol/L Tris-HCl (pH 7.5), 50 mmol/L NaCl, 3 mmol/L MgCl2, 1 mmol/L DTT, and 0.05% CHAPS] containing 10 μg/mL PI substrate (l-α-phosphatidylinositol; Avanti Polar Lipids; prepared in 3% octyl-glucoside) and the PI3K proteins (10, 25, 10, and 150 nmol/L of p110α, p110β, p110δ, and p110γ, respectively) were then added. The reaction was started by the addition of 5 μL of 1 μmol/L ATP prepared in the reaction buffer and ran for either 60 (for p110α, p110β, and p110δ) or 120 min (for p110γ) and subsequently terminated by the addition of 10 μL Kinase-Glo buffer (Promega). The plates were then read in a Synergy 2 reader (BioTek) for luminescence detection.

Cell lines

Human prostate tumor cell line PC3M, U87MG glioblastoma tumor line

Preparation method

Soluble in DMSO. General tips for obtaining a higher concentration: Please warm the tube at 37 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reacting condition

10 to 50 nmol/L, 30 min

Applications

In the U87MG glioblastoma PTEN-negative cell line, NVP-BEZ235 reduced S473P-Akt and T308P-Akt levels in a dose-dependent manner. Treatment of U2OS cells stably expressing a GFP-FKHRL1 chimeric protein with NVP-BEZ235 led to its complete nuclear relocalization. In PTEN-null cell lines PC3M and U87MG, NVP-BEZ235 dose-dependently reduced cell proliferation with an average GI50 of 10 to 12 nmol/L. In PC3M cells, NVP-BEZ235 (10-50 nmol/L) increased the G1 population. NVP-BEZ235 significantly increased the amount of the cyclin-dependent kinase inhibitor p27Kip1 in the p53-negative PC3M cell line.

Animal models

PC3M tumor-bearing nude mice, U87MG tumor-bearing mice

Dosage form

Oral administration, 50 mg/kg daily or 25 mg/kg twice daily

Application

NVP-BEZ235 (50 mg/kg) appeared rapidly in plasma with a Cmax of 1.68 μmol/L at 0.5 h and a C24h of 0.03 μmol/L. In U87MG tumor-bearing mice, suboptimal (25 mg/kg/d once per day) to optimal (45 mg/kg/d once per day) of NVP-BEZ235 caused regression of the tumors. NVP-BEZ235 caused disease stasis when administered orally as a single agent and could enhance the efficacy of other anticancer agents when used in in vivo combination studies.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

BEZ235 is an imidazoquinoline derivative inhibiting both PI3K and mTOR kinases with low nanomolar IC50s. It was well tolerated in preclinical animal studies as well as in clinical trials with manageable gastrointestinal side-effects^{[1, 2]}. It competes with ATP by binding to the ATP-binding site of kinases and reversibly reduces enzyme activity, resulting in growth arrest of tumor cells in G1 phase^[1]. Besides the inhibition of cell growth, BEZ235 blocks VEGF-induced angiogenesis^[3]. It may also inhibit DNA-PKcs^[4].

BEZ235 has shown potential anti-tumor activity both in vitro and in vivo. It inhibited growth of multiple cancer cell lines independently of mutation status in PI3K pathway^[5]. In xenograft mice models, it blocked PI3K signaling and showed antitumor activity^{[1, 5]}. Combination study demonstrated that it enhances the efficacy of temozolomide^[1].

Clinical data shows anti-tumor activity of BEZ235 treatment, especially in cancer patients with deregulated PI3K signaling pathway. This compound is currently under investigation in multiple clinical trials either as monotherapy or in combination with other agents.

References:
1. Maira SM, Stauffer F, Brueggen J et al. Identification and characterization of NVP-BEZ235, a new orally available dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor with potent in vivo antitumor activity. Mol Cancer Ther 2008; 7: 1851-1863.
2. Markman B, Tabernero J, Krop I et al. Phase I safety, pharmacokinetic, and pharmacodynamic study of the oral phosphatidylinositol-3-kinase and mTOR inhibitor BGT226 in patients with advanced solid tumors. Ann Oncol 2012; 23: 2399-2408.
3. Schnell CR, Stauffer F, Allegrini PR et al. Effects of the dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235 on the tumor vasculature: implications for clinical imaging. Cancer Res 2008; 68: 6598-6607.
4. Mukherjee B, Tomimatsu N, Amancherla K et al. The dual PI3K/mTOR inhibitor NVP-BEZ235 is a potent inhibitor of ATM- and DNA-PKCs-mediated DNA damage responses. Neoplasia 2012; 14: 34-43.
5. Serra V, Markman B, Scaltriti M et al. NVP-BEZ235, a dual PI3K/mTOR inhibitor, prevents PI3K signaling and inhibits the growth of cancer cells with activating PI3K mutations. Cancer Res 2008; 68: 8022-8030.