Conservation of structure, function and inhibitor binding in UNC-51-like kinase 1 and 2 (ULK1/2).

Autophagy is essential for cellular homeostasis and when deregulated this survival mechanism has been associated with disease development. Inhibition of autophagy initiation by inhibiting the kinase ULK1 (Unc-51-like autophagy activating kinase 1) has been proposed as a potential cancer therapy. While inhibitors and crystal structures of ULK1 have been reported, little is known about the other closely related kinase ULK2 (Unc-51-like autophagy activating kinase 2). Here, we present the crystal structure of ULK2 in complex with ATP competitive inhibitors. Surprisingly, the ULK2 structure revealed a dimeric assembly reminiscent of dimeric arrangements of auto-activating kinases suggesting a role for this association in ULK activation. Screening of a kinase focused library of pre-clinical and clinical compounds revealed several potent ULK1/2 inhibitors and good correlation of inhibitor-binding behavior with both ULK kinases. Aurora A was identified as a major off-target of currently used ULK1 inhibitors. Autophagic flux assays demonstrated that this off-target activity by strongly inducing autophagy in different cellular systems conferred an additional layer of complexity in the interpretation of cellular data. The data presented here provide structural models and chemical starting points for the development of ULK1/2 dual inhibitors with improved selectivity for future exploitation of autophagy inhibition.

[1]  The Duy Nguyen,et al.  Loss of the selective autophagy receptor p62 impairs murine myeloid leukemia progression and mitophagy. , 2019, Blood.

[2]  Jeffrey P. MacKeigan,et al.  A Potent and Selective ULK1 Inhibitor Suppresses Autophagy and Sensitizes Cancer Cells to Nutrient Stress , 2018, iScience.

[3]  Hong-Gang Wang,et al.  Targeted Inhibition of ULK1 Promotes Apoptosis and Suppresses Tumor Growth and Metastasis in Neuroblastoma , 2018, Molecular Cancer Therapeutics.

[4]  I. Dikic,et al.  Mechanism and medical implications of mammalian autophagy , 2018, Nature reviews. Molecular cell biology.

[5]  N. Mizushima.  A brief history of autophagy from cell biology to physiology and disease , 2018, Nature Cell Biology.

[6]  S. Fulda.  Targeting autophagy for the treatment of cancer , 2018, Biological chemistry.

[7]  Christopher J. Williams,et al.  MolProbity: More and better reference data for improved all‐atom structure validation , 2018, Protein science : a publication of the Protein Society.

[8]  A. Thorburn,et al.  Targeting autophagy in cancer , 2017, Nature Reviews Cancer.

[9]  Owen Johnson,et al.  Integrating macromolecular X-ray diffraction data with the graphical user interface iMosflm , 2017, Nature Protocols.

[10]  R. Amaravadi,et al.  Targeting Autophagy in Cancer: Update on Clinical Trials and Novel Inhibitors , 2017, International journal of molecular sciences.

[11]  Saijuan Chen,et al.  Caspase-3 controls AML1-ETO-driven leukemogenesis via autophagy modulation in a ULK1-dependent manner. , 2017, Blood.

[12]  G. J. Yoshida.  Therapeutic strategies of drug repositioning targeting autophagy to induce cancer cell death: from pathophysiology to treatment , 2017, Journal of Hematology & Oncology.

[13]  Paul Emsley,et al.  Tools for ligand validation in Coot , 2017, Acta crystallographica. Section D, Structural biology.

[14]  Airlie J McCoy,et al.  Acknowledging Errors: Advanced Molecular Replacement with Phaser. , 2017, Methods in molecular biology.

[15]  N. Mizushima,et al.  An Autophagic Flux Probe that Releases an Internal Control. , 2016, Molecular cell.

[16]  Chang S. Chan,et al.  Autophagy provides metabolic substrates to maintain energy charge and nucleotide pools in Ras-driven lung cancer cells , 2016, Genes & development.

[17]  Z. Ye,et al.  ULK1/2 Constitute a Bifurcate Node Controlling Glucose Metabolic Fluxes in Addition to Autophagy. , 2016, Molecular cell.

[18]  J. Yong,et al.  Uncoordinated 51‐like kinase 2 signaling pathway regulates epithelial‐mesenchymal transition in A549 lung cancer cells , 2016, FEBS letters.

[19]  Mary G. Lin,et al.  Structure and function of the ULK1 complex in autophagy. , 2016, Current opinion in cell biology.

[20]  Gregory McAllister,et al.  Macroautophagy is dispensable for growth of KRAS mutant tumors and chloroquine efficacy , 2015, Proceedings of the National Academy of Sciences.

[21]  I. Ganley,et al.  Pharmacological inhibition of ULK1 kinase blocks mammalian target of rapamycin (mTOR)-dependent autophagy. , 2015, The Journal of Biological Chemistry.

[22]  K. Shokat,et al.  Discovery and structure of a new inhibitor scaffold of the autophagy initiating kinase ULK1. , 2015, Bioorganic & medicinal chemistry.

[23]  John M Asara,et al.  Small Molecule Inhibition of the Autophagy Kinase ULK1 and Identification of ULK1 Substrates. , 2015, Molecular cell.

[24]  I. Ganley,et al.  Pharmacological Inhibition of ULK1 Kinase Blocks Mammalian Target of Rapamycin (mTOR)-dependent Autophagy* , 2015, The Journal of Biological Chemistry.

[25]  K. Shokat,et al.  Structure of the Human Autophagy Initiating Kinase ULK1 in Complex with Potent Inhibitors , 2014, ACS chemical biology.

[26]  Y. Murakami,et al.  Inhibition of autophagy induces retinal pigment epithelial cell damage by the lipofuscin fluorophore A2E , 2014, FEBS open bio.

[27]  A. Thorburn,et al.  Autophagy and Cancer Therapy , 2014, Molecular Pharmacology.

[28]  Chang S. Chan,et al.  Autophagy is required for glucose homeostasis and lung tumor maintenance. , 2014, Cancer discovery.

[29]  Ruedi Aebersold,et al.  Early Steps in Autophagy Depend on Direct Phosphorylation of Atg9 by the Atg1 Kinase , 2014, Molecular cell.

[30]  P. Sykacek,et al.  A dual role for autophagy in a murine model of lung cancer , 2014, Nature Communications.

[31]  D. Bernlohr,et al.  Distinct functions of Ulk1 and Ulk2 in the regulation of lipid metabolism in adipocytes , 2013, Autophagy.

[32]  T. Jacks,et al.  Autophagy suppresses progression of K-ras-induced lung tumors to oncocytomas and maintains lipid homeostasis. , 2013, Genes & development.

[33]  Philip R. Evans,et al.  How good are my data and what is the resolution? , 2013, Acta crystallographica. Section D, Biological crystallography.

[34]  D. Klionsky,et al.  Autophagic Processes in Yeast: Mechanism, Machinery and Regulation , 2013, Genetics.

[35]  A. Harris,et al.  Transcriptional up-regulation of ULK1 by ATF4 contributes to cancer cell survival. , 2013, The Biochemical journal.

[36]  Xuejun Jiang,et al.  The ULK1 complex , 2013, Autophagy.

[37]  S. Tooze,et al.  Regulation of nutrient-sensitive autophagy by uncoordinated 51-like kinases 1 and 2 , 2013, Autophagy.

[38]  A. Morgan,et al.  Association Analysis of ULK1 with Crohn's Disease in a New Zealand Population , 2012, Gastroenterology research and practice.

[39]  Stefan Knapp,et al.  Kinase inhibitor selectivity profiling using differential scanning fluorimetry. , 2012, Methods in molecular biology.

[40]  Masaaki Komatsu,et al.  Autophagy: Renovation of Cells and Tissues , 2011, Cell.

[41]  C. Tournier,et al.  The requirement of uncoordinated 51-like kinase 1 (ULK1) and ULK2 in the regulation of autophagy , 2011, Autophagy.

[42]  C. Thompson,et al.  Ammonia-induced autophagy is independent of ULK1/ULK2 kinases , 2011, Proceedings of the National Academy of Sciences.

[43]  H. Coller,et al.  Activated Ras requires autophagy to maintain oxidative metabolism and tumorigenesis. , 2011, Genes & development.

[44]  Eileen White,et al.  Autophagy and Metabolism , 2010, Science.

[45]  C. James,et al.  Akt and Autophagy Cooperate to Promote Survival of Drug-Resistant Glioma , 2010, Science Signaling.

[46]  S. Gygi,et al.  Network organization of the human autophagy system , 2010, Nature.

[47]  W. Kabsch.  XDS , 2010, Acta crystallographica. Section D, Biological crystallography.

[48]  E. Masliah,et al.  Beclin 1 Gene Transfer Activates Autophagy and Ameliorates the Neurodegenerative Pathology in α-Synuclein Models of Parkinson's and Lewy Body Diseases , 2009, The Journal of Neuroscience.

[49]  S. Tooze,et al.  Evolution of Atg1 function and regulation , 2009, Autophagy.

[50]  She Chen,et al.  ULK1·ATG13·FIP200 Complex Mediates mTOR Signaling and Is Essential for Autophagy* , 2009, Journal of Biological Chemistry.

[51]  Sarah L. Brown,et al.  A key role for autophagy and the autophagy gene Atg16l1 in mouse and human intestinal Paneth cells , 2008, Nature.

[52]  Daniel J. Klionsky,et al.  Autophagy fights disease through cellular self-digestion , 2008, Nature.

[53]  Stefan Knapp,et al.  Activation segment dimerization: a mechanism for kinase autophosphorylation of non-consensus sites , 2008, The EMBO journal.

[54]  S. Knapp,et al.  A systematic interaction map of validated kinase inhibitors with Ser/Thr kinases , 2007, Proceedings of the National Academy of Sciences.

[55]  S. Tooze,et al.  siRNA Screening of the Kinome Identifies ULK1 as a Multidomain Modulator of Autophagy* , 2007, Journal of Biological Chemistry.

[56]  V. Karantza,et al.  Role of Autophagy in Breast Cancer , 2007, Autophagy.

[57]  L. Pearl,et al.  Activation segment exchange: a common mechanism of kinase autophosphorylation? , 2007, Trends in biochemical sciences.

[58]  C. Figueroa,et al.  UNC-51-like kinase regulation of fibroblast growth factor receptor substrate 2/3. , 2007, Cellular signalling.

[59]  Sibylle Mittnacht,et al.  Trans‐activation of the DNA‐damage signalling protein kinase Chk2 by T‐loop exchange , 2006, The EMBO journal.

[60]  Philippe Dessen,et al.  Inhibition of Macroautophagy Triggers Apoptosis , 2005, Molecular and Cellular Biology.

[61]  Pavol Skubák,et al.  Direct incorporation of experimental phase information in model refinement. , 2004, Acta crystallographica. Section D, Biological crystallography.

[62]  H. Hibshoosh,et al.  Induction of autophagy and inhibition of tumorigenesis by beclin 1 , 1999, Nature.