Figure 3.
Phase 2: Characterization of the specific actives and mechanism-of-action studies. Specific actives (126 compounds) were further tested in a series of cell-based, biochemical, and biophysical assays to identify potential mechanisms of action. Cell viability assays using KRAS-dependent and -independent human colorectal cancer cell lines were performed. Cell-based assays [pERK, pAKT, and pEGFR homogeneous time-resolved fluorescence (HTRF)] as well as biochemical assays for BRAFV600E, MEK1, ERK2, phosphoinositide 3-kinase alpha (PI3Kα), AKT1, and mammalian target of rapamycin (mTOR) activities were performed to assess RAS-related pathway activities. Biophysical assays were also performed to assess direct compound KRASWT and KRASG12D binding.