Application of isothermal titration calorimetry and column chromatography for identification of biomolecular targets

Area of Science:

• Biochemistry
• Analytical Chemistry
• Pharmacology

Background:

• Identifying drug targets in complex biomolecular mixtures is crucial for drug discovery.
• Traditional methods for target identification can be limited by the need for labeled or modified molecules.
• Unknown or uncharacterized ligands and targets pose significant challenges in biochemical research.

Purpose of the Study:

• To present a robust protocol for identifying unknown target proteins interacting with a given drug or lead compound.
• To demonstrate the utility of isothermal titration calorimetry (ITC) as a tracking tool in target identification.
• To provide a method applicable to targets and ligands that are not amenable to labeling or modification.

Main Methods:

• Combines chromatography for fractionation of biomolecular mixtures with isothermal titration calorimetry (ITC) for binding verification.
• ITC is initially used to confirm ligand binding within the mixture.
• Iterative chromatographic purification of fractions, guided by ITC, leads to target protein identification.

Main Results:

• Successfully identifies both low-abundance and highly abundant target proteins from complex mixtures.
• The protocol has been validated by internal teams and external researchers.
• Target protein identification is achievable within a 3-5 day timeframe.

Conclusions:

• This combined chromatography-ITC approach offers an effective strategy for identifying unknown drug targets.
• The method is particularly valuable when dealing with unlabeled or unmodified biomolecules.
• The protocol provides a rapid and reliable solution for target deconvolution in biochemical and pharmacological studies.