title: Affinity classification of kinase inhibitors by mass spectrometric methods and validation using standard IC(50) measurements.
abstract: Protein kinases have emerged as a major drug target in the last years. Since more than 500 kinases are encoded in the human genome, cross-reactivity of a majority of kinase inhibitors causes problems. Tools are required for a rapid classification of inhibitors according to their affinity for a certain target to refine the search for new, more specific lead compounds. Mass spectrometry (MS) is increasingly used in pharmaceutical research and drug discovery to investigate protein-ligand interactions and determination of binding affinities. We present a comparison of different existing nanoelectrospray-MS based methods to quantify binding affinities and qualitatively rank, by competitive experiments, the affinity of several clinical inhibitors. We also present a new competitive method which is derived from our previous work for quantitative assessment of binding strengths (Wortmann et al., J. Mass Spectrom. 2008, 43(5), 600-608). The human kinases studied for this purpose were p38alpha (MAPK14) and LCK (lymphocyte specific kinase), and their interaction with 17 known small molecule kinase inhibitors was probed. Moreover, we present a new method to differentiate type I from type II inhibitors (Liu, Y.; Gray, N. S. Nat. Chem. Biol. 2006, 2(7), 358-364) based on a kinetic experiment with direct MS read-out of the noncovalent complex between the human kinase and the inhibitor. This method was successfully applied to p38alpha binding to BIRB796, as well as to a BIRB796 analogue. Quantitative determination of the binding strength is also described. The results of our competitive experiments for the affinity classification of different inhibitors, as well as the results for the kinetic study, are in good agreement with IC(50) measurements and data found in the literature.
authors.affiliation: Department of Chemistry and Applied Biosciences, ETH Zurich, CH-8093 Zürich, Switzerland.
authors.forename: Matthias Conradin
authors.initials: MC
authors.lastname: Jecklin
authors.forename: David
authors.initials: D
authors.lastname: Touboul
authors.forename: Rishi
authors.initials: R
authors.lastname: Jain
authors.forename: Estee Naggar
authors.initials: EN
authors.lastname: Toole
authors.forename: John
authors.initials: J
authors.lastname: Tallarico
authors.forename: Peter
authors.initials: P
authors.lastname: Drueckes
authors.forename: Paul
authors.initials: P
authors.lastname: Ramage
authors.forename: Renato
authors.initials: R
authors.lastname: Zenobi
mesh_terms: D001667:Binding, Competitive; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000037:antagonists & inhibitors* / Q000737:chemistry; D048308:Mitogen-Activated Protein Kinase 14 / Q000037:antagonists & inhibitors* / Q000737:chemistry; D036103:Nanotechnology / Q000295:instrumentation / Q000379:methods; D047428:Protein Kinase Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D040901:Proteomics / Q000295:instrumentation / Q000379:methods; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*
chemical_list: D047428:Protein Kinase Inhibitors; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D048308:Mitogen-Activated Protein Kinase 14