Affinity characterization-mass spectrometry methodology for quantitative analyses of small molecule protein binding...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Pharmacology

Background:

• Macromolecule-ligand interactions are crucial in biological processes and drug discovery.
• Accurate determination of binding affinity is essential for understanding these interactions.
• Existing methods may face limitations in directly quantifying binding parameters.

Purpose of the Study:

• To introduce and validate Affinity Characterization by Mass Spectrometry (AC-MS) as a novel quantitative methodology.
• To demonstrate AC-MS's ability to directly determine all equilibrium binding concentration terms.
• To assess the utility of AC-MS for drug discovery studies.

Main Methods:

• Utilized Liquid Chromatography-Mass Spectrometry (LC-MS) for quantitative analysis.
• Employed AC-MS to measure macromolecule (M), ligand (L), and complex (ML) concentrations.
• Performed saturation analyses to determine dissociation constants and binding stoichiometry.

Main Results:

• AC-MS directly determined all three concentration terms (M, L, ML) of the binding equation.
• Obtained accurate dissociation constants and binding modes for DNA primase and thymidylate synthetase interactions.
• Data were consistent with established thermal-denaturation techniques and literature inhibition constants.
• Demonstrated AC-MS applicability to various soluble protein-ligand systems.

Conclusions:

• AC-MS is a robust method for quantitative affinity characterization of small molecule ligands.
• The technique provides direct measurement of binding parameters and solubility information.
• AC-MS serves as a valuable tool for drug discovery, particularly for counter screening and structure-activity relationship studies.