title: Development of an HTS-Compatible Assay for the Discovery of Ulk1 Inhibitors.
abstract: A rapidly accumulating body of work suggests the autophagy pathway is an attractive therapeutic target for neurodegenerative diseases and cancer. To validate autophagy as an anticancer strategy and to assess if systemic inhibition of the pathway will have deleterious effects on normal tissues and physiology, highly selective autophagy inhibitors are needed. While several inducers and inhibitors of autophagy are known, all are nonspecific and none target the enzymes that execute the pathway. A central upstream regulator of the autophagy pathway is the serine/threonine kinase Ulk1 (UNC-51-like kinase-1). Selective molecular probes that function as Ulk1-specific inhibitors are needed to improve our understanding of the autophagy pathway. To identify inhibitors of Ulk1 kinase activity, we developed an HTS-compatible, homogeneous biochemical assay using AlphaScreen technology. This novel assay design uses purified stress-activated Ulk1 and monitors phosphorylation of its full-length native substrate, Atg13. This assay was optimized and validated in a 384-well format by screening the Sigma LOPAC library. Here we report that the Ulk1 AlphaScreen assay is robust and reproducible, with a Z' factor value of 0.83 ± 0.02 and a signal to background ratio of 20 ± 1.2. Thus, this assay can be used to screen large chemical libraries to discover novel inhibitors of Ulk1.
authors: Department of Molecular Therapeutics, Scripps Florida, Jupiter, FL, USA.
authors: Laura H
authors: LH
authors: Rosenberg
authors: Department of Molecular Therapeutics, Scripps Florida, Jupiter, FL, USA.
authors: Marie
authors: M
authors: Lafitte
authors: Department of Molecular Therapeutics, Scripps Florida, Jupiter, FL, USA.
authors: Wayne
authors: W
authors: Grant
authors: Department of Molecular Therapeutics, Scripps Florida, Jupiter, FL, USA.
authors: Weimin
authors: W
authors: Chen
authors: Department of Tumor Biology, The Moffitt Cancer Center and Research Institute, Tampa, FL, USA.
authors: John L
authors: JL
authors: Cleveland
authors: Department of Molecular Therapeutics, Scripps Florida, Jupiter, FL, USA ducketdr@scripps.edu.
authors: Derek R
authors: DR
authors: Duckett
mesh_terms: D002460:Cell Line; D055808:Drug Discovery / Q000379:methods*; D004354:Drug Screening Assays, Antitumor / Q000379:methods; D057166:High-Throughput Screening Assays; D006801:Humans; D007700:Kinetics; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases
keywords: cancer and cancer drugs; kinases; oncology; signal transduction