In vitro activity: LYN-1604 is a ULK1 agonist. There are 3 amino acids (LYS50, LEU53, and TYR89) that are key to the activation site of LYN-1604 and ULK1 by site-directed mutagenesis and biochemical assays. LYN-1604 induces cell death involved in ATF3, RAD21, and caspase3, accompanied by autophagy and apoptosis. LYN-1604 is bound to wild-type ULK1 with a binding affinity in the nanomole range (KD = 291.4 nM), but the ULK1Y89A mutant protein causes a sharp decrease in binding affinity with lower response and Kd than wild-type ULK1, ULK1K50A and ULK1L53A mutants

Kinase Assay: Cells or tumor tissues are treated with LYN-1604 for the indicated times. Both adherent and floating cells are collected and western blot analysis is carried out. Briefly, the cell pellets are resuspended with lysis buffer consisting of 50 mM HEPES, pH 7.4; 1% Triton-X-100, 2 mM sodium orthovanadate, 100 mM sodium fluoride, 1 mM edetic acid, 1 mM PMSF, 10 mg/L aprotinin and 10 mg/Lleupeptin, and lysed at 4°C for 1 h. After 12 000 rpm centrifugation for 15 min, the protein content of the supernatant is determined by the Bio-Rad DC protein assay.

Cell Assay: Cells are dispensed in 96-well plates at a density of 5 × 104 cells per mL. After 24 h of incubation, cells are treated with different concentrations of compounds for the indicated time periods. Cell viability is measured by the MTT assay.