In vitro activity: (S)-crizotinib disrupts nucleotide pool homeostasis via MTH1 inhibition, induces an increase in DNA single-strand breaks, and activates DNA repair in human colon carcinoma cells.

Kinase Assay: Half-maximal inhibitory concentrations (IC50) are determined using a luminescence-based assay with some minor modifications. Briefly, serial dilutions of compounds are dissolved in assay buffer (100 mM Tris-acetate pH 7.5, 40 mM NaCl and 10 mM Mg(OAc)2 containing 0.005% Tween-20 and 2 mM dithiothreitol (DTT). Upon addition of MTH1 recombinant protein (final concentration 2 nM), plates are incubated on a plate shaker for 15 min at room temperature. After addition of the substrate dGTP (final concentration 100 μM), 8-oxo-dGTP (final concentration 13.2 μM), or 2-OH-dATP (final concentration 8.3 μM) the generation of pyrophosphate (PPi) as a result of nucleotide triphosphate hydrolysis by MTH1 is monitored over a time course of 15 min using the PPi Light Inorganic Pyrophosphate Assay kit. IC50 values are determined by fitting a dose-response curve to the data points using nonlinear regression analysis using the GraphPad Prism software.

Cell Assay: One day before treatment, cells are seeded per well in six-well plates and incubated for 24 h. The next day DMSO (equal to highest amount of compound dilution, maximum 0.2%) or compounds in increasing concentrations were added and cells incubated at 37 °C, 5% CO2, for 7-10 days. After washing with PBS, cells are fixed with ice-cold methanol, stained with crystal violet solution (0.5% in 25% methanol) and left to dry overnight. For quantification of results, ultraviolet absorbance of crystal violet is determined at 595 nm following solubilisation by 70% ethanol. Data are analysed using nonlinear regression analysis using the GraphPad Prism software.

Nature. 2014 Apr 10;508(7495):222-7.