ITC
Functional Application Areas
Application Notes
Isothermal Titration Calorimetry and Drug Design
Calorimetry Takes the Heat Off Drug Discovery
Making Cool Drugs Hot: The Use of Isothermal Titration Calorimetry as a Tool to Study Binding Energetics
Revealing Kinase Inhibitor Mechanisms: ITC Leads the Way
Divided We Fall? Studying Low Affinity Fragments of Ligands by ITC
In the Mix: Simultaneous Determination for Isomers and Enantiomers
Related Links
ITC of Ligand Mixtures - Reverse Titrations
Mathematica Workbook for Simultaneous Determination for Isomers and Enantiomers
Measurement of Tight Binding Affinities with ITC
Binding Studies with Differential Scanning Calorimetry (DSC)
Protein-Small Molecule Interactions
Many proteins in biological systems have specific binding sites for small molecules, and these binding events are critical to the protein’s activity and function. Proteins include enzymes, antibodies, receptors, transport proteins, membrane proteins, cytokines and hormones. These small molecules, often called “ligands” include but are not limited to:
- Drugs
- Metals
- Sugars
- Nucleotides
- Fatty acids
- Cofactors
- Cell signaling molecules
- Surfactants
- Enzyme substrates
- Enzyme inhibitors
Knowledge of these protein-small molecule interactions is important to understand how proteins function in biological systems. There have been rapid advances in structural biology and relating structure to biochemical function and mechanism. However, knowledge of protein structure alone does not ensure accurate prediction of function and biological activity. The complete characterization of any binding interaction requires a quantification of the affinity, number of binding sites, and the thermodynamics.
Thermodynamic data, specifically enthalpy (ΔH) and entropy (ΔS), reveal the forces that drive complex formation and mechanism of action. Thermodynamics provide information on conformational changes, hydrogen bonding, hydrophobic interactions, and charge-charge interactions. This information is used to describe the function and mechanism at a molecular level.
Isothermal Titration Calorimetry (ITC) is a powerful analytical tool which measures the binding affinity and thermodynamics between any two biomolecules. ITC is considered the “gold standard” assay for binding.
ITC is vital in the study of multi-probe structure activity relationship (SAR) since it can detect contributions that affinity-only methods may miss. For example, the affinity measured by these methods may be similar for a wild-type and mutant protein binding to a drug, but ITC can reveal differences in ΔH and ΔS that can describe the mechanism of action of binding. This information can validate in-silico modeling. ITC is also commonly used to validate other binding assays.
Since ITC is done in-solution, it can utilize any biological buffer. For a full characterization of a biomolecular interaction, it is important to observe how salt, pH, temperature, etc affects binding affinity and thermodynamics.
Most drug targets are proteins, and drug discovery involves identifying compounds which can either inhibit or activate the target protein. ITC is also becoming an important tool in characterizing drug-target interactions, and can be used in many different stages of Drug Discovery and Development. ITC is also used to characterize ligand specificity (ie a series of metals binding to the same site of a protein), binding of competitive inhibitors, and studying allosteric effects.
References
Isothermal titration calorimetry: experimental design, data analysis, and probing macromolecule/ligand binding and kinetic interactions.
Freyer M. W. and Lewis E. A.
Methods Cell Biol 84, 79-113 (2008)
Isothermal titration calorimetry and differential scanning calorimetry as complementary tools to investigate the energetics of biomolecular recognition.
Jelesarov I. and Bosshard H. R.
J Mol Recognit 12, 3-18 (1999)
Microcalorimetry: a response to challenges in modern biotechnology.
Krell, T.
Microbial Biotechnol 1, 126-136 (2008)
Application of isothermal titration calorimetry in the biological sciences: things are heating up!
Ladbury J. E.
Biotechniques 37, 885-887 (2004)
Direct measurement of protein binding energetics by isothermal titration calorimetry.
Leavitt S. and Freire E.
Curr Opin Struct Biol 11, 560-566 (2001)
Applications of Biocalorimetry: Binding, stability and enzyme kinetics
O’Brien R. and Haq I.
in Biocalorimetry 2: Applications of Calorimetry in the Biological Sciences. Ladbury, J.E., Doyle, M.L., eds., John Wiley & Sons Ltd., Chichester UK, pp. 3-34 (2004)
Isothermal titration calorimetry of biomolecules
O’Brien R., Ladbury J.E., and Chowdhry B.Z.
in Protein-Ligand Interactions: Hydrodynamics and Calorimetry. Harding, S.E., Chowdhry, B.Z., eds., Oxford University Press, Oxford UK, pp. 263-286 (2001
ITC in the post-genomic era...? Priceless.
Velazquez-Campoy A. and Freire E.
Biophys Chem 115, 115-124 (2005)
Applications of calorimetric methods to drug discovery and the study of protein interactions.
Weber P. C. and Salemme F. R.
Curr Opin Struct Biol 13, 115-121 (2003)
ITC – Protein-Small Molecule Interactions Reference List
ITC – Drug Discovery and Design Reference List
ITC – Protein Engineering and Mutagenesis Reference List
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