title: Kinobead and Single-Shot LC-MS Profiling Identifies Selective PKD Inhibitors.
abstract: ATP-competitive protein kinase inhibitors are important research tools and therapeutic agents. Because there are >500 human kinases that contain highly conserved active sites, the development of selective inhibitors is extremely challenging. Methods to rapidly and efficiently profile kinase inhibitor targets in cell lysates are urgently needed to discover selective compounds and to elucidate the mechanisms of action for polypharmacological inhibitors. Here, we describe a protocol for microgram-scale chemoproteomic profiling of ATP-competitive kinase inhibitors using kinobeads. We employed a gel-free in situ digestion protocol coupled to nanoflow liquid chromatography-mass spectrometry to profile ∼200 kinases in single analytical runs using as little as 5 μL of kinobeads and 300 μg of protein. With our kinobead reagents, we obtained broad coverage of the kinome, monitoring the relative expression levels of 312 kinases in a diverse panel of 11 cancer cell lines. Further, we profiled a set of pyrrolopyrimidine- and pyrazolopyrimidine-based kinase inhibitors in competition-binding experiments with label-free quantification, leading to the discovery of a novel selective and potent inhibitor of protein kinase D (PKD) 1, 2, and 3. Our protocol is useful for rapid and sensitive profiling of kinase expression levels and ATP-competitive kinase inhibitor selectivity in native proteomes.
authors.affiliation: Department of Pharmacology, School of Medicine and Department of Chemistry, University of Washington , Seattle, Washington 98195, United States.
authors.forename: Martin
authors.identifier: 0000-0002-0996-1655
authors.initials: M
authors.lastname: Golkowski
authors.affiliation: Department of Pharmacology, School of Medicine and Department of Chemistry, University of Washington , Seattle, Washington 98195, United States.
authors.forename: Rama Subba Rao
authors.initials: RS
authors.lastname: Vidadala
authors.affiliation: Department of Pharmacology, School of Medicine and Department of Chemistry, University of Washington , Seattle, Washington 98195, United States.
authors.forename: Chloe K
authors.initials: CK
authors.lastname: Lombard
authors.affiliation: Department of Pharmacology, School of Medicine and Department of Chemistry, University of Washington , Seattle, Washington 98195, United States.
authors.forename: Hyong Won
authors.initials: HW
authors.lastname: Suh
authors.affiliation: Department of Pharmacology, School of Medicine and Department of Chemistry, University of Washington , Seattle, Washington 98195, United States.
authors.forename: Dustin J
authors.initials: DJ
authors.lastname: Maly
authors.affiliation: Department of Pharmacology, School of Medicine and Department of Chemistry, University of Washington , Seattle, Washington 98195, United States.
authors.forename: Shao-En
authors.initials: SE
authors.lastname: Ong
mesh_terms: D000970:Antineoplastic Agents; D001667:Binding, Competitive; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D047428:Protein Kinase Inhibitors / Q000032:analysis*; D011494:Protein Kinases / Q000032:analysis; D040901:Proteomics / Q000379:methods*; D011743:Pyrimidines / Q000494:pharmacology / Q000627:therapeutic use; D011758:Pyrroles / Q000494:pharmacology / Q000627:therapeutic use; D013347:Subcellular Fractions / Q000737:chemistry
chemical_list: D000970:Antineoplastic Agents; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D011758:Pyrroles; C527741:pyrrolopyrimidine; D011494:Protein Kinases; C089212:protein kinase D; D011493:Protein Kinase C
keywords: SILAC; kinase inhibitor; kinobeads; protein kinase D; proteomics; target identification