title: Small Molecule Inhibition of the Autophagy Kinase ULK1 and Identification of ULK1 Substrates.
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.
authors.affiliation: Molecular and Cell Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA.
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Egan
authors.affiliation: Molecular and Cell Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA.
authors.forename: Matthew G H
authors.initials: MG
authors.lastname: Chun
authors.affiliation: Cell Death and Survival Networks Research Program, NCI-Designated Cancer Center, Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Mitchell
authors.initials: M
authors.lastname: Vamos
authors.affiliation: Cell Death and Survival Networks Research Program, NCI-Designated Cancer Center, Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Haixia
authors.initials: H
authors.lastname: Zou
authors.affiliation: Cell Death and Survival Networks Research Program, NCI-Designated Cancer Center, Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Juan
authors.initials: J
authors.lastname: Rong
authors.affiliation: Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520, USA.
authors.forename: Chad J
authors.initials: CJ
authors.lastname: Miller
authors.affiliation: Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520, USA.
authors.forename: Hua Jane
authors.initials: HJ
authors.lastname: Lou
authors.affiliation: Cell Death and Survival Networks Research Program, NCI-Designated Cancer Center, Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Dhanya
authors.initials: D
authors.lastname: Raveendra-Panickar
authors.affiliation: Functional Genomics Core, Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Chih-Cheng
authors.initials: CC
authors.lastname: Yang
authors.affiliation: Cell Death and Survival Networks Research Program, NCI-Designated Cancer Center, Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Sheffler
authors.affiliation: Cell Death and Survival Networks Research Program, NCI-Designated Cancer Center, Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Peter
authors.initials: P
authors.lastname: Teriete
authors.affiliation: Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA 02115, USA; Department of Medicine, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: John M
authors.initials: JM
authors.lastname: Asara
authors.affiliation: Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520, USA.
authors.forename: Benjamin E
authors.initials: BE
authors.lastname: Turk
authors.affiliation: Cell Death and Survival Networks Research Program, NCI-Designated Cancer Center, Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA. Electronic address: ncosford@sanfordburnham.org.
authors.forename: Nicholas D P
authors.initials: ND
authors.lastname: Cosford
authors.affiliation: Molecular and Cell Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA; Howard Hughes Medical Institute, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA. Electronic address: shaw@salk.edu.
authors.forename: Reuben J
authors.initials: RJ
authors.lastname: Shaw
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001343:Autophagy / Q000187:drug effects / Q000502:physiology*; D000071189:Autophagy-Related Protein-1 Homolog; D001549:Benzamides / Q000737:chemistry / Q000494:pharmacology*; D020134:Catalytic Domain / Q000235:genetics; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D016384:Consensus Sequence; D055786:Gene Knockout Techniques; D057809:HEK293 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D011743:Pyrimidines / Q000737:chemistry / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001549:Benzamides; D047908:Intracellular Signaling Peptides and Proteins; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; C000601952:SBI-0206965; D000071189:Autophagy-Related Protein-1 Homolog; D017346:Protein Serine-Threonine Kinases; C484754:ULK1 protein, human; C112444:Ulk1 protein, mouse