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Fluorophore labeling of the glycine-rich loop as a method of identifying inhibitors that bind to active and inactive kinase conformations.

Simard JR, Getlik M, Grütter C, Schneider R, Wulfert S, Rauh D
J Am Chem Soc 132 4152-60 (2010)
Related entries: 3hub, 3huc

Cited: 29 times
EuropePMC logo PMID: 20201574

Abstract

Targeting protein kinases with small organic molecules is a promising strategy to regulate unwanted kinase activity in both chemical biology and medicinal chemistry research. Traditionally, kinase inhibitors are identified in activity-based screening assays using enzymatically active kinase preparations to measure the perturbation of substrate phosphorylation, often resulting in the enrichment of classical ATP competitive (Type I) inhibitors. However, addressing enzymatically incompetent kinase conformations offers new opportunities for targeted therapies and is moving to the forefront of kinase inhibitor research. Here we report the development of a new FLiK (Fluorescent Labels in Kinases) binding assay to detect small molecules that induce changes in the conformation of the glycine-rich loop. Due to cross-talk between the glycine-rich loop and the activation loop in kinases, this alternative labeling approach can also detect ligands that stabilize inactive kinase conformations, including slow-binding Type II and Type III kinase inhibitors. Protein X-ray crystallography validated the assay results and identified a novel DFG-out binding mode for a quinazoline-based inhibitor in p38alpha kinase. We also detected the high-affinity binding of a clinically relevant and specific VEGFR2 inhibitor, and we provide structural details of its binding mode in p38alpha, in which it stabilizes the DFG-out conformation. Last, we demonstrate the power of this new FLiK labeling strategy to detect the binding of Type I ligands that induce conformational changes in the glycine-rich loop as a means of gaining affinity for the target kinase. This approach may be a useful alternative to develop direct binding assays for kinases that do not adopt the DFG-out conformation while also avoiding the use of expensive kits, detection reagents, or radioactivity frequently employed with activity-based assays.

Articles - 3l8s mentioned but not cited (6)

  1. Comprehensive Modeling and Discovery of Mebendazole as a Novel TRAF2- and NCK-interacting Kinase Inhibitor. Tan Z, Chen L, Zhang S. Sci Rep 6 33534 (2016)
  2. Convolutional neural network scoring and minimization in the D3R 2017 community challenge. Sunseri J, King JE, Francoeur PG, Koes DR, Koes DR. J Comput Aided Mol Des 33 19-34 (2019)
  3. p38α Mitogen-Activated Protein Kinase Is a Druggable Target in Pancreatic Adenocarcinoma. Yang L, Sun X, Ye Y, Lu Y, Zuo J, Liu W, Elcock A, Zhu S. Front Oncol 9 1294 (2019)
  4. Fragment-Based Structural Optimization of a Natural Product Itampolin A as a p38α Inhibitor for Lung Cancer. Liang JW, Wang MY, Wang S, Li XY, Meng FH. Mar Drugs 17 (2019)
  5. In Silico Screening and In Vitro Activity Measurement of Javamide Analogues as Potential p38 MAPK Inhibitors. Park JB. Int J Mol Sci 18 E2704 (2017)
  6. Structural basis of a redox-dependent conformational switch that regulates the stress kinase p38α. Pous J, Baginski B, Martin-Malpartida P, González L, Scarpa M, Aragon E, Ruiz L, Mees RA, Iglesias-Fernández J, Orozco M, Nebreda AR, Macias MJ. Nat Commun 14 7920 (2023)


Reviews citing this publication (4)

  1. Exploration of type II binding mode: A privileged approach for kinase inhibitor focused drug discovery? Zhao Z, Wu H, Wang L, Liu Y, Knapp S, Liu Q, Gray NS. ACS Chem Biol 9 1230-1241 (2014)
  2. Fluorescent biosensors for high throughput screening of protein kinase inhibitors. Prével C, Pellerano M, Van TN, Morris MC. Biotechnol J 9 253-265 (2014)
  3. Fluorescent Reporters and Biosensors for Probing the Dynamic Behavior of Protein Kinases. González-Vera JA, Morris MC. Proteomes 3 369-410 (2015)
  4. A Comprehensive Structural Overview of p38α MAPK in Complex with Type I Inhibitors. Astolfi A, Iraci N, Manfroni G, Barreca ML, Cecchetti V. ChemMedChem 10 957-969 (2015)

Articles citing this publication (19)

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  3. DFG-out mode of inhibition by an irreversible type-1 inhibitor capable of overcoming gate-keeper mutations in FGF receptors. Huang Z, Tan L, Wang H, Liu Y, Blais S, Deng J, Neubert TA, Gray NS, Li X, Mohammadi M. ACS Chem Biol 10 299-309 (2015)
  4. Overcoming EGFRG724S-mediated osimertinib resistance through unique binding characteristics of second-generation EGFR inhibitors. Fassunke J, Müller F, Keul M, Michels S, Dammert MA, Schmitt A, Plenker D, Lategahn J, Heydt C, Brägelmann J, Tumbrink HL, Alber Y, Klein S, Heimsoeth A, Dahmen I, Fischer RN, Scheffler M, Ihle MA, Priesner V, Scheel AH, Wagener S, Kron A, Frank K, Garbert K, Persigehl T, Püsken M, Haneder S, Schaaf B, Rodermann E, Engel-Riedel W, Felip E, Smit EF, Merkelbach-Bruse S, Reinhardt HC, Kast SM, Wolf J, Rauh D, Büttner R, Sos ML. Nat Commun 9 4655 (2018)
  5. Using small molecules to target protein phosphatases. Vintonyak VV, Waldmann H, Rauh D. Bioorg Med Chem 19 2145-2155 (2011)
  6. Structure-based discovery of the first allosteric inhibitors of cyclin-dependent kinase 2. Rastelli G, Anighoro A, Chripkova M, Carrassa L, Broggini M. Cell Cycle 13 2296-2305 (2014)
  7. Insights into MAPK p38alpha DFG flip mechanism by accelerated molecular dynamics. Filomia F, De Rienzo F, Menziani MC. Bioorg Med Chem 18 6805-6812 (2010)
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  10. Neuritogenic militarinone-inspired 4-hydroxypyridones target the stress pathway kinase MAP4K4. Schröder P, Förster T, Kleine S, Becker C, Richters A, Ziegler S, Rauh D, Kumar K, Waldmann H. Angew Chem Int Ed Engl 54 12398-12403 (2015)
  11. Virtual screening using a conformationally flexible target protein: models for ligand binding to p38α MAPK. Vinh NB, Simpson JS, Scammells PJ, Chalmers DK. J Comput Aided Mol Des 26 409-423 (2012)
  12. Structural and chemical insights into the covalent-allosteric inhibition of the protein kinase Akt. Uhlenbrock N, Smith S, Weisner J, Landel I, Lindemann M, Le TA, Hardick J, Gontla R, Scheinpflug R, Czodrowski P, Janning P, Depta L, Quambusch L, Müller MP, Engels B, Rauh D. Chem Sci 10 3573-3585 (2019)
  13. FGFR1 Kinase Inhibitors: Close Regioisomers Adopt Divergent Binding Modes and Display Distinct Biophysical Signatures. Klein T, Tucker J, Holdgate GA, Norman RA, Breeze AL. ACS Med Chem Lett 5 166-171 (2014)
  14. Role of Molecular Interactions and Protein Rearrangement in the Dissociation Kinetics of p38α MAP Kinase Type-I/II/III Inhibitors. You W, Chang CA. J Chem Inf Model 58 968-981 (2018)
  15. p38α MAPK and Type I Inhibitors: Binding Site Analysis and Use of Target Ensembles in Virtual Screening. Astolfi A, Iraci N, Sabatini S, Barreca ML, Cecchetti V. Molecules 20 15842-15861 (2015)
  16. Discovery of Potent c-MET Inhibitors with New Scaffold Having Different Quinazoline, Pyridine and Tetrahydro-Pyridothienopyrimidine Headgroups. Jiang Y, Zhang K, Gao S, Wang G, Huang J, Wang J, Chen L. Molecules 21 (2016)
  17. A Bioorganometallic Approach to Study Histidine Kinase Autophosphorylations. Wang N, She Z, Ingar Z, Martic S, Kraatz HB. Chemistry 23 3152-3158 (2017)
  18. Chromophore carbonyl twisting in fluorescent biosensors encodes direct readout of protein conformations with multicolor switching. Allert MJ, Kumar S, Wang Y, Beese LS, Hellinga HW. Commun Chem 6 168 (2023)
  19. Structure-based design, synthesis and crystallization of 2-arylquinazolines as lipid pocket ligands of p38α MAPK. Bührmann M, Wiedemann BM, Müller MP, Hardick J, Ecke M, Rauh D. PLoS One 12 e0184627 (2017)


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