TGX-221, an effective, specific, and cell membrane permeable inhibitor of the PI3K p110β catalytic subunit, is utilized for cancer treatment.

p110β:5 nM, p110δ:0.1 μM

In mouse models, TGX-221 has been shown to enhance blood flow, prolonging tail bleeding and renal bleeding time.

In J774.2 macrophages, TGX-221 inhibits the phosphorylation of Ser473 on PKB induced by insulin. It also impedes platelet-ECC (extracorporeal circulation) interactions, platelet aggregation, and the binding between platelets and granulocytes in an ECC model. Furthermore, in the PC3 cells, TGX-221 (at concentrations of 0.2-20 μM) can suppress cell proliferation and reduce the activity of the p110β subunit of PI3K.

Lipid kinase activity : IC50 values are measured using a standard lipid kinase activity with PI as a substrate. (i)100 μM cold ATP is used instead of 10 μM, (ii) the DMSO concentration is 1%, and (iii) [γ-33P]ATP is used instead of [γ-32P]ATP. The TLC plates are quantified using a phosphorimager screen. The reported IC50 values are determined by non-linear regression analysis on the basis of at least three independent experiments repeated across multiple preparations of recombinant protein.

For measurement of proliferation, cells are seeded in triplicate in 96-well culture plates and incubated overnight to allow cell attachment. The cells are incubated with TGX-221 for 24, 48, and 72 hours. At designated time intervals, cells are quantified by a crystal violet staining-based colorimetric assay. Briefly, cells are fixed by addition of 100 μl of 2.5% glutaraldehyde solution and incubated at room temperature for 30 minutes. Plates are washed three times by submersion in PBS solution. Plates are air-dried and stained by addition of 100 μL of 0.1% solution of crystal violet dissolved in deionized water and incubated for 20 minutes at room temperature, excess dye is removed by extensive washing with deionized water, and plates are air-dried prior to bound dye solubilization in 100 μL of 10% acetic acid. The optical density of dye extracts is measured directly in plates using a microplate reader at 570 nm.(Only for Reference)