Figure 4.

XST-14 is a potent ULK1 kinase inhibitor that binds to the KD of ULK1. (A) Chemical structure and chemical synthesis route of XST-14, a ULK1 competitive kinase inhibitor. (B) The kinetic interactions of XST-14 and the ULK1 protein were determined by surface plasmon resonance (SPR) analyses. The indicated concentrations of XST-14 were passed over immobilized ULK1 on CM5 sensor chips. The affinity constants were evaluated using BIAevaluation software. (C) The kinetic interactions of XST-14 and the KD truncation of the ULK1 protein were determined by SPR analyses. The indicated concentrations of XST-14 were passed over immobilized KD on streptavidin (SA) sensor chips. The affinity constants were evaluated using BIAevaluation software. (D-F) The kinetic interactions of the mutated KD and XST-14 were determined by SPR analyses. The indicated concentrations of XST-14 were passed over the immobilized mutants of the KD on CM5 sensor chips. The affinity constants were evaluated using BIAevaluation software. (G) The half maximal inhibitory concentration (IC₅₀) for XST-14 against ULK1 was determined using an in vitro kinase assay. XST-14 was tested in triplicate in a 12-dose IC₅₀ mode with a starting dose of 100 μM. The data are the means ± SEM. (H, I) The kinase selectivity profile for XST-14 was determined using SelectScreen Kinase Profiling Services. Briefly, XST-14 was screened at a 5 μM dose for its ability to impair the binding of a panel of 403 kinases to the substrate in an in vitro binding assay. The red box indicates >75% inhibition. (J) The summary of IC₅₀ measurements for each kinase is presented in this table.