High-throughput small-molecule inhibitor screening

Cell lines were screened for sensitivity against a panel of 104 small-molecule inhibitors as previously described.¹⁷ Briefly, cells were exposed to graded concentrations of each drug in 384 well plates with a seeding density of 250 cells per well in 50 μL final volume per well. Each drug was tested across an eight-point, threefold interval dose–response curve (including the no-drug control). After 3 days, relative abundance of viable cells was quantified in each well using a tetrazolium-based MTS assay. All absorbance values from the MTS assay were normalised to the average of 48 wells per plate containing no drug, and these normalised values were used to fit a third-order polynomial curve to each drug dose–response. IC₅₀ values were interpreted from this curve fit to assess the relative sensitivity of each cell line to each drug.