Small Molecule Inhibition of the Autophagy Kinase ULK1 and Identification of ULK1 Substrates

Mol Cell. 2015 Jul 16;59(2):285-97. doi: 10.1016/j.molcel.2015.05.031. Epub 2015 Jun 25.

Abstract

Many tumors become addicted to autophagy for survival, suggesting inhibition of autophagy as a potential broadly applicable cancer therapy. ULK1/Atg1 is the only serine/threonine kinase in the core autophagy pathway and thus represents an excellent drug target. Despite recent advances in the understanding of ULK1 activation by nutrient deprivation, how ULK1 promotes autophagy remains poorly understood. Here, we screened degenerate peptide libraries to deduce the optimal ULK1 substrate motif and discovered 15 phosphorylation sites in core autophagy proteins that were verified as in vivo ULK1 targets. We utilized these ULK1 substrates to perform a cell-based screen to identify and characterize a potent ULK1 small molecule inhibitor. The compound SBI-0206965 is a highly selective ULK1 kinase inhibitor in vitro and suppressed ULK1-mediated phosphorylation events in cells, regulating autophagy and cell survival. SBI-0206965 greatly synergized with mechanistic target of rapamycin (mTOR) inhibitors to kill tumor cells, providing a strong rationale for their combined use in the clinic.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Autophagy / drug effects
  • Autophagy / physiology*
  • Autophagy-Related Protein-1 Homolog
  • Benzamides / chemistry
  • Benzamides / pharmacology*
  • Catalytic Domain / genetics
  • Cell Survival / drug effects
  • Cell Survival / physiology
  • Cells, Cultured
  • Consensus Sequence
  • Gene Knockout Techniques
  • HEK293 Cells
  • Humans
  • Intracellular Signaling Peptides and Proteins / antagonists & inhibitors*
  • Intracellular Signaling Peptides and Proteins / genetics
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Mice
  • Molecular Sequence Data
  • Phosphorylation
  • Protein Kinase Inhibitors / chemistry
  • Protein Kinase Inhibitors / pharmacology*
  • Protein-Serine-Threonine Kinases / antagonists & inhibitors*
  • Protein-Serine-Threonine Kinases / deficiency
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism*
  • Pyrimidines / chemistry
  • Pyrimidines / pharmacology*
  • RNA, Small Interfering / genetics
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Substrate Specificity

Substances

  • Benzamides
  • Intracellular Signaling Peptides and Proteins
  • Protein Kinase Inhibitors
  • Pyrimidines
  • RNA, Small Interfering
  • Recombinant Proteins
  • SBI-0206965
  • Autophagy-Related Protein-1 Homolog
  • Protein-Serine-Threonine Kinases
  • ULK1 protein, human
  • Ulk1 protein, mouse