In vitro activity: CGK733 is able to confer robust growth to senescent cells that have ceased proliferation. Senescence-associated β-galactosidase (SA–β-gal) activity disappears in CGK733-treated cells. CGK733 shows greater potency in inhibiting ATM/ATR than LY294002 (IC50 , ~5 μM for ATM and ATR), a pan-inhibitor of PI3K and PIKKs. CGK733 (30 μM) treated for 24h causes ~60% cell death in senescent MCF-7 cells. CGK733 (20 μM) induces the loss of cyclin D1 via the ubiquitin- dependent proteasomal degradation pathway in MCF-7 and T47D breast cancer cell lines. CGK733 at concentrations ranging from 0.6- 40 μM, inhibits proliferation of MCF-7 and T47D estrogen receptor (ER) positive breast cancer cells, MDA-MB436 ER negative breast cancer cells, LnCap pros-tate cancer cells and HCT116 colon cancer cells. Furthermore, CGK733 also suppresses proliferation of non- transformed mouse BALB/c 3T3 embryonic fibroblast cells. The CGK733-mediated inhibition of proliferation is dose dependent and significant at doses as low as 2.5 μM.

Cell Assay: Cells are seeded in 96-well plates at a predetermined, optimal cell density to ensure exponential growth for duration of the assay. After a 24 h preincubation, growth medium is replaced with experimental medium containing the appropriate drug concentrations or 0.1% (v/v) vehicle control. After a 48 h incubation, cell proliferation is estimated using the sulforhodamine B colorimetric assay.