Microscale Thermophoresis for AURKA Binding

Microscale thermophoresis (MST) was performed for assessment of the interaction between compound (5) and AURKA (Sigma-Aldrich, Germany). The method is performed as previously described [31 (link), 32 (link)]. The AURKA protein was labeled using Monolith Protein Labeling Kit RED- NHS 2nd Generation (MO-L011, NanoTemper Technologies GmbH, Munich, Germany) according to manufacturer’s instructions. The AURKA protein concentration used was 400 nm, it was titrated against different concentrations of compound (5). Analysis buffer used includes; 50 mM Tris buffer pH 7.0, 150 mM NaCl, 10 mM MgCl₂ and 0.05% Tween 20. Samples of interaction were filled into Monolith NT.115 standard capillaries (MO-K022, NanoTemper Technologies GmbH, Munich, Germany). Monolith NT.115 instrument (NanoTemper Technologies) was used for fluorescent signal measurement. Test was performed using 60% LED power and 40 MST power. For analysis, we used MO.Affinity analysis software (Nano Temper Technologies) to generate of fitting curve of interaction and calculation of dissociation constant (Kd).