2.7. Kinase Kd Determination

IQ-1L was submitted for dissociation constant (K_d) determination using KINOMEscan (Eurofins Pharma Discovery, San Diego, CA, USA), as described previously [55]. In brief, kinases were produced and displayed on T7 phage or expressed in HEK-293 cells. Binding reactions were performed at room temperature for 1 h, and the fraction of kinase not bound to the test compound was determined by capture with an immobilized affinity ligand and quantified by quantitative polymerase chain reaction. Primary screening at fixed concentrations of compound was performed in duplicate. For dissociation constant K_d determination, a 12-point half-log dilution series (a maximum concentration of 33 μM) was used. Assays were performed in duplicate, and their average mean value is displayed.