3.4. Surface Plasmon Resonance (SPR) Analysis

The expression and purification of DCLK1 kinase domain were same as those described in the Supplementary Materials in our previous work [7]. Briefly, DCLK1 kinase domain was overexpressed in Escherichia coli strain Rosetta (DE3) (Novagen, Darmstadt, Germany) and purified sequentially by nickel affinity chromatography and gel filtration chromatography. SPR experiments were performed using a Biacore X-100 system (Cytiva, Washington, DC, USA) at room temperature, with a running buffer of 10 mM Na₂HPO₄, 1.75 mM KH₂PO₄, pH 7.2~7.6, supplemented with 0.137 M NaCl, 2.65 mM KCl, 0.05% Tween-20, and 5% DMSO. DCLK1 kinase domain (KD) was covalently immobilized onto a CM5 sensor chip using the Amine coupling kit (GE Healthcare). The immobilization of KD resulted in ~12,000 response units (RU). Small-molecule inhibitors were injected over the flow cells at a range of eight concentrations prepared by serial two-fold dilutions, at a flow rate of 30 μL min⁻¹, with an association time of 120 s and a dissociation time of 180 s. The data were fitted to a 1:1 binding model so as to calculate equilibrium dissociation constants (binding affinity, K_D) using GraphPad Prism 8.