Microscale thermophoresis (MST) assay for in vitro binding affinity

Recombinant human MTH1 was labeled with the Monolith NT™ Protein Labeling Kit RED (Cat#L001) according to the supplied labeling protocol [32]. Labelled MTH1 was kept constant at 100 nM, while all samples tested were diluted in a 20 mM HEPES (pH 7.5) and 0.05 (v/v) % Tween-20. Compounds were diluted in 12 dilution steps covering the range from 500 μM to 200 nM. After 10 min incubation at room temperature about 37°C, samples were loaded into Monolith™ standard-treated capillaries and the thermophoresis was measured at 25°C after 30 min incubation on a Monolith NT.115 instrument (NanoTemper Technologies, München, Germany). Laser power was set to 20% using 30 seconds on-time. The LED power was set to 100%. The dissociation constant Kd values were fitted by using the NTAnalysis software (NanoTemper Technologies, München, Germany). The Kd value of the binding was obtained in two ways. The first way was calculated by thermophoresis curve when there was no fluorescent change during the initial fluorescence scanning. The second way was calculated from the initial fluorescence scanning curve when compounds bound in the sites which directly interfered the fluorescence. The appropriate way was employed to obtain Kd values of eight compounds and (S)–crizotinib.