Kinase inhibitor library screen and IC50 determination

The cell lines SNU-878, SNU-886, SNU-398, CAL-72, and PEER were screened for kinase inhibitors using a kinase inhibitor-focused library (LINCS). The LINCS library contained 197 kinase inhibitors, from a diverse ATP competitive kinase inhibitor set. These kinase inhibitors were shown to be relatively potent and selective towards a narrow range of targets. 2000 adherent cells and 3000 suspension cells were plated with 50 μL medium in each well of a 384 microplate. Drugs with a concentration of 660 nM were added the same day. After 48 hours cellular proliferation was determined using CellTiter-Glo (Promega). Drugs identified as having a significant effect on this screen were tested in greater detail. 2000 adherent or 4000 suspension cells were plated on day 0 on a 96 well plate with 100 μL medium, except on the marginal wells. Drugs were added on day 1, which were serially diluted three-fold from 10 μM to 1.5 nM. Cell viability was determined after 72 hours of treatment by adding 20 μL CellTiter-Glo. Cell viability was determined using XLfit4.0 software. IC₅₀ values were calculated using Graphpad Prism as the drug concentration that reduced cell viability by 50% compared to untreated cells. Rapamycin was purchased from LC laboratories and ganetespib from Synta Pharmaceuticals.