title: Thermodynamic signatures of fragment binding: Validation of direct versus displacement ITC titrations.
abstract: BACKGROUND
  Detailed characterization of the thermodynamic signature of weak binding fragments to proteins is essential to support the decision making process which fragments to take further for the hit-to-lead optimization.
  
  
  METHODS
  Isothermal titration calorimetry (ITC) is the method of choice to record thermodynamic data, however, weak binding ligands such as fragments require the development of meaningful and reliable measuring protocols as usually sigmoidal titration curves are hardly possible to record due to limited solubility.
  
  
  RESULTS
  Fragments can be titrated either directly under low c-value conditions (no sigmoidal curve) or indirectly by use of a strong binding ligand displacing the pre-incubated weak fragment from the protein. The determination of Gibbs free energy is reliable and rather independent of the applied titration protocol.
  
  
  CONCLUSIONS
  Even though the displacement method achieves higher accuracy, the obtained enthalpy-entropy profile depends on the properties of the used displacement ligand. The relative enthalpy differences across different displacement experiments reveal a constant signature and can serve as a thermodynamic fingerprint for fragments. Low c-value titrations are only reliable if the final concentration of the fragment in the sample cell exceeds 2-10 fold its K(D) value. Limited solubility often prevents this strategy.
  
  
  CONCLUSIONS
  The present study suggests an applicable protocol to characterize the thermodynamic signature of protein-fragment binding. It shows however, that such measurements are limited by protein and fragment solubility. Deviating profiles obtained by use of different displacement ligands indicate that changes in the solvation pattern and protein dynamics most likely take influence on the resulting overall binding signature.
authors.affiliation: Institute of Pharmaceutical Chemistry, Philipps-University Marburg, Marbacher Weg 6, 35037 Marburg, Germany.
authors.forename: Eggert
authors.initials: E
authors.lastname: Rühmann
authors.affiliation: Institute of Pharmaceutical Chemistry, Philipps-University Marburg, Marbacher Weg 6, 35037 Marburg, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Betz
authors.affiliation: Bayer HealthCare, Müllerstr. 178, 13353 Berlin, Germany.
authors.forename: Marie
authors.initials: M
authors.lastname: Fricke
authors.affiliation: Institute of Pharmaceutical Chemistry, Philipps-University Marburg, Marbacher Weg 6, 35037 Marburg, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Heine
authors.affiliation: Bayer HealthCare, Müllerstr. 178, 13353 Berlin, Germany.
authors.forename: Martina
authors.initials: M
authors.lastname: Schäfer
authors.affiliation: Institute of Pharmaceutical Chemistry, Philipps-University Marburg, Marbacher Weg 6, 35037 Marburg, Germany.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Klebe
mesh_terms: D002151:Calorimetry / Q000379:methods*; D011485:Protein Binding; D013816:Thermodynamics
keywords: Displacement titrations; Isothermal titration calorimetry; Low-c value titration; Protein–fragment binding; Thermodynamic signature; Thrombin