| Description: |
BEZ235 is a dual pan-class I PI3K and mTOR kinase inhibitor with IC50s of 4 nM/5 nM/7 nM/75 nM, and 20.7 nM for p110α/p110γ/p110δ/p110β and mTOR, respectively. BEZ235 inhibits both mTORC1 and mTORC2. |
| In Vivo: |
BEZ235 (NVP-BEZ235) (45 mg/kg, p.o.) treatment induces colonic tumor regression in a GEM model for sporadic PIK3CA wild-type CRC[2]. NVP-BEZ235 (45 mg/kg) is administered to MENX rats (n=2 each group) by oral gavage and animals are sacrificed 1 or 6 hours after treatment. Immunostains for P-AKT and P-S6 show considerable reduction of the two proteins, and particularly of P-S6, 6 hours after administration of NVP-BEZ235 when compares with PEG-treated rats. At 6 hours after treatment, the pituitary adenomas of NVP-BEZ235-treated rats has a proteomic profile significantly different from the tumors of placebo-treated rats[3]. |
| In Vitro: |
BEZ235 (NVP-BEZ235) potently inhibits PI3K in an ATP Competitive Manner. NVP-BEZ235 (250 nM) significantly reduced the phosphorylation levels of the mTOR activated kinase p70S6K. NVP-BEZ235 also leads to a reduction of S235/S236P-RPS6 levels with an IC50 of 6.5 nM, suggesting that NVP-BEZ235 can directly inhibit the mTOR kinase, as the kinase domain of mTOR is highly homologous to the one of class IA PI3K. The activity of NVP-BEZ235 against mTOR is confirmed using a biochemical mTOR K-LISA assay (IC50, 20.7 nM)[1]. The IC50s of NVP-BEZ235 for HCT116, DLD-1, and SW480 cell lines are 14.3±6.4, 9.0±1.5, and 12.0±1.6 nM, respectively[2]. |
| Kinase Assay: |
PI3Kα, β, and δ proteins are composed of the iSH2 domain of p85 NH2-terminally fused to the full-length protein p110 protein, with the exception of α that also does not contain the last 20 amino acids. PI3Kγ is produced as full-length protein deleted for its first 144 amino acids. All constructs are 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 are then cotransfected with BaculoGold WT genomic DNA using methods recommended by the vendor for production of the respective recombinant baculoviruses and proteins. Compounds are tested for their activity against PI3K using a Kinase-Glo assay. The kinase reaction is done in 384-well black plate. Each well is loaded with 50 nL of test items (in 90% DMSO) and 5 μL reaction buffer [10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 3 mM MgCl2, 1 mM 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 nM of p110α, p110β, p110δ, and p110γ, respectively) are then added. The reaction is started by the addition of 5 μL of 1 μM 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. The plates are then read in a Synergy 2 reader for luminescence detection[1]. |
| Cell Assay: |
HCT116 (PIK3CA mutant; kinase domain at H1047R), DLD-1 (PIK3CA mutant; helical domain at E545K), and SW480 (PIK3CA wild-type) human CRC cell lines (ATCC) and isogenic DLD-1 PIK3CA mutant and wild-type cells are maintained in DMEM with 10% FBS and 1× Penicillin/Streptomycin. Cells are plated at different initial densities (HCT116: 3,000 cells/well, DLD-1: 5,500 cells/well, SW480: 4,500 cells/well, DLD-1 PIK3CA mutant: 7,000 cells/well, and DLD-1 PIK3CA wild-type: 9,000 cells/well) to account for differential growth kinetics. After 16 hours, cells are incubated with increasing concentrations of BEZ235 (10, 100, 1000 nM), and drug-containing growth medium is changed every 24 hours. Cell viability is assessed 16 hours after the initial plating and 48 hours after initiation of drug treatment using the colorimetric MTS assay CellTiter 96 AQueous One Solution Cell Proliferation Assay. Cell viability after drug treatment is normalized to that of untreated cells also grown for 48 hours. IC50 values are calculated using 4 parameter nonlinear regression in GraphPad Prism 5[2]. |
| Animal Administration: |
Mice[2] Tumor-bearing Apc CKO mice are randomly assigned to treatment with either control vehicle alone (n=8) or 45 mg/kg body weight BEZ235 in 10% 1-methyl-2-pyrrolidone/90% PEG 300 (n=8) by daily oral gavage for 28 days. The treatment dose is chosen based on literature indicating that 40-50 mg/kg body weight BEZ235 effectively treats murine tumor models without adverse effects. Base on pharmacokinetic studies demonstrating maximal tissue concentration one hour after NVP-BEZ235 administration, tumor-bearing mice are sacrificed one hour after final treatment dose. Colonic tumor volume is assessed using calipers (width×length×height) and tumors are harvested for both western blot analysis and immunohistochemistry. Rats[3] MENX-affected rats used. Three doses of BEZ235 are tested in MENX rats: 20, 30, and 45 mg/kg. As the two higher doses causes a weight loss >10% after 10 days of treatment, the dose of 20 mg/kg is used for further studies. For MRI studies, MENX-affected rats at 7 to 8 months of age (with sizeable adenomas but still in good general health) are treated for 14 days with BEZ235 (20 mg/kg) or placebo (PEG) administered daily per oral gavage. |
| References: |
[1]. Maira SM, 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(7), 1851-1863.
[2]. Roper J, et al. The dual PI3K/mTOR inhibitor NVP-BEZ235 induces tumor regression in a genetically engineered mouse model of PIK3CA wild-type colorectal cancer. PLoS One, 2011, 6(9), e25132.
[3]. Lee M, et al. Targeting PI3K/mTOR Signaling Displays Potent Antitumor Efficacy against Nonfunctioning Pituitary Adenomas. Clin Cancer Res. 2015 Jul 15;21(14):3204-15. |
