Affinity Classification of Kinase Inhibitors by Mass Spectrometric Methods and Validation Using Standard IC50 Measurements

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Author(s) Jecklin, Matthias Conradin, Touboul, David, Jain, Rishi, Toole, Estee Naggar, Tallarico, John, Drueckes, Peter, Ramage, Paul, Zenobi, Renato
Publication Type Journal Items, Publication Status: Published
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Title Affinity Classification of Kinase Inhibitors by Mass Spectrometric Methods and Validation Using Standard IC50 Measurements
Author(s) Jecklin, Matthias Conradin
Touboul, David
Jain, Rishi
Toole, Estee Naggar
Tallarico, John
Drueckes, Peter
Ramage, Paul
Zenobi, Renato
Journal or Series Title Analytical chemistry
Volume Number 81
Issue Number 1
Start Page 408
End Page 419
ISSN 0003-2700
Publisher American Chemical Society
Publication Place Washington, DC
Publication Date 2009
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 p38α (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 p38α 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 IC50 measurements and data found in the literature.
DOI 10.1021/ac801782c
Additional Notes Received 25 August 2008, Accepted 3 November 2008, Published online 9 December 2008
Document Type Article
Publication Status Published
Language English
Assigned Organisational Unit(s) 03430
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NEBIS System Number 000005465
Source Database ID PP-49089
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  author = "Jecklin, Matthias Conradin and Touboul, David and Jain, Rishi and Toole, Estee Naggar and Tallarico, John and Drueckes, Peter and Ramage, Paul and Zenobi, Renato",
  title = "{A}ffinity {C}lassification of {K}inase {I}nhibitors by {M}ass {S}pectrometric {M}ethods and {V}alidation {U}sing {S}tandard {I}{C}50 {M}easurements",
  journal = "Analytical chemistry",
  year = 2009,
  volume = "81",
  number = "1",
  pages = "408--419",

E-Citations record created: Fri, 02 Apr 2010, 03:35:20 CET