eCF309

To verify that eCF309 inhibits both mTORC1 and mTORC2 signalling in cells by inhibition of the mTOR catalytic domain, phosphorylation of downstream targets of both complexes were assessed in MCF7 breast cancer cells by Western blot. Phosphorylation levels of the p70 ribosomal S6 kinase 1 (P70S6K) and its substrate S6 were analysed as substrates of mTORC1, and phospho-AKT (Y473) as an mTORC2 substrate. eCF309 mediated a dose-dependent reduction of the pP70S6K, pS6 and pAKT levels in MCF7 cells, achieving almost complete inhibition of the phosphorylation of these targets at 30 nM. Antiproliferative activity of eCF309 against cancer cell lines was also evaluated using the PrestoBlue reagent. EC50 = 8 nM in MCF7 cells, 37 nM in PC3 cells; 72 nM in MDA-MB-231 cells. As expected by an mTOR inhibitor, eCF309 reduced cell proliferation in these cell lines by cell cycle arrest.