Isothermal Titration Calorimetry (ITC) is a quantitative technique for studying biomolecular interactions, measuring the heat released or absorbed during binding events, which allows for the determination of binding constants, stoichiometry, enthalpy, and entropy in a single experiment without modifications to the molecules. ITC can elucidate the mechanisms behind molecular interactions, aiding in drug discovery, candidate selection, and optimization. While ITC has advantages such as providing a comprehensive thermodynamic profile and being a non-destructive method, it also has drawbacks, including a requirement for larger sample sizes and slower throughput.

1. Isothermal Titration Calorimetry (ITC)
Pharmaceutical Analysis
T. Y B. Pharm
By
Prof. Khemchand R. Surana
(M. S. Pharm)
Divine College of Pharmacy, Satana,
Nashik
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2. Isothermal Titration
Calorimetry (ITC)
• Isothermal Titration Calorimetry (ITC) is a technique
used in quantitative studies of a wide variety of
biomolecular interactions.
•A direct measurement of heat generated or absorbed
when molecules interact.
•It works by directly measuring the heat that is either
released or absorbed during a biomolecular binding
event.
•ITC is the only technique that can simultaneously
determine all binding parameters in a single experiment.
•Requiring no modification of binding partners, either with
fluorescent tags or through immobilization, ITC measures2KRS-Divine

3. Cont…..
•Measuring heat transfer during binding enables
accurate determination of binding constants (KD),
reaction stoichiometry (n), enthalpy (∆H) and entropy
(ΔS).
•This provides a complete thermodynamic profile of the
molecular interaction.
•ITC goes beyond binding affinities and can elucidate
the mechanisms underlying molecular interactions.
•This deeper understanding of structure-function
relationships enables more confident decision making in
hit selection and lead optimization.
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5. Measurement Principle
•Isothermal Titration Calorimetry is used to measure
reactions between biomolecules.
•The methodology allows determination of the binding
affinity, stoichiometry, and entropy and enthalpy of the
binding reaction in solution, without the need to use
labels.
•When binding occurs, heat is either absorbed or
released and this is measured by the sensitive
calorimeter during gradual titration of the ligand into
the sample cell containing the biomolecule of interest.
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6. How it works…
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7. Working …
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10. Instrumentation
The thermal core:
•In the microcalorimeter there are two cells, one of which
contains water and acts as a reference cell, the other
contains the sample.
•The microcalorimeter needs to keep these two cells at
exactly the same temperature.
•The heat sensing devices detect temperature difference
between the cells when binding occurs and give feedback
to the heaters, which compensate for this difference and
return the cells to equal temperature.
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11. Instrumentation
Making a measurement:
•The reference cell and the sample cell are set to the
desired experimental temperature.
•The ligand is loaded into a syringe which sits in a very
accurate injection device.
•The injection device is inserted into the sample cell
containing the protein of interest.
•A series of small aliquots of ligand are injected into the
protein solution.
• If there is a binding of the ligand to the protein, heat
changes of a few millionths of a degree Celsius are
detected and measured.
•As the first injection is made, the microcalorimeter
measures all heat released until the binding reaction has
reached equilibrium. 11KRS-Divine

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18. Advantages of ITC
In comparison with other biophysical techniques, the key
advantages are:
•Complete basic thermodynamic characterization
(stoichiometry, association constant, and binding enthalpy)
in a single experiment.
•Heat is a universal signal, and, hence, no need for reporter
labels (e.g. chromophores, fluorophores).
•Direct determination of the binding enthalpy.
•Non-destructive technique.
•Interaction in solution
•Possibility of performing experiment with optically dense
solutions or unusual systems (e.g. dispersions, intact
organelles or cells)
•Considerably fast. 18KRS-Divine

19. Disadvantages of ITC
There are however, certain disadvantages:
•Signal is proportional to the binding enthalpy, and the
non-covalent complexes may exhibit rather small
binding enthalpies
•Heat is a universal signal and each process
contributes to the global measured heat, thus
complicating the evaluation of the contribution because
of binding
•Large amount of sample is needed, though this has
come down considerably
•It is a slow technique with a low throughput (0.25 - 2
h/assay), not suitable for HTS
•Kinetically slow processes may be overlooked
•A limited range for consistently measured binding
affinities.
•Before now, no kinetic information for binding
interactions could be accessed 19KRS-Divine

20. Applications
ITC is widely used in drug discovery and
development for:
•Quantify binding affinity
•Candidate selection and optimization
•Measurement of thermodynamics and active
concentration
•Characterization of mechanism of action
•Confirmation of intended binding targets in small
molecule drug discovery
•Determination of binding specificity and
stoichiometry
•Validation of IC50 and EC50 values during hit-to- 20KRS-Divine

21. Online video link
https://www.malvernpanalytical.com/en/pr
oducts/technology/microcalorimetry/isothe
rmal-titration-calorimetry
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