Molecular dynamics simulations provide insights into ULK-101 potency and selectivity toward autophagic kinases ULK1/2.

Kinase domains are highly conserved within proteins in both sequence and structure. Many factors, including phosphorylation, amino acid substitutions or mutations, and small molecule inhibitor binding, influence conformations of the kinase domain and enzymatic activity. ULK1 and ULK2 are serine/threonine kinases that serve important roles in autophagy, an intracellular recycling process capable of degrading proteins and organelles fusion with lysosomes. ULK1/2 are emerging as therapeutic targets in human cancer, particularly KRAS-driven malignancies. Here, we performed molecular dynamics (MD) simulations to hypothesize bound poses for the ULK1/2 small molecule inhibitor, ULK-101. We observed stable bound states for ULK-101 to the adenosine triphosphate (ATP)-binding site of ULK2, coordinated by hydrogen bonding with the hinge backbone and the catalytic lysine sidechain. Notably, ULK-101 occupies a hydrophobic pocket associated with the N-terminus of the αC-helix. Large movements in the phosphate-binding loop (P-loop) are also associated with ULK-101 inhibitor binding and exit from ULK2. Together, our data support a model to explain ULK-101 potency toward ULK1/2.