Improved Rapid Equilibrium Dialysis-Mass Spectrometry (RED-MS) Method for Measuring Small Molecule-Protein Complex...

Area of Science:

• Biochemistry
• Pharmacology
• Analytical Chemistry

Background:

• Rapid equilibrium dialysis (RED) is a standard technique for assessing drug absorption, distribution, metabolism, and excretion (ADME) properties.
• Characterizing small molecule binding affinities to proteins is crucial for drug discovery and development.
• Traditional methods can be challenging for large protein complexes or when analyzing numerous compounds.

Purpose of the Study:

• To introduce and validate an improved Rapid Equilibrium Dialysis method coupled with Mass Spectrometry (RED-MS).
• To enable robust and high-throughput measurement of small molecule binding affinities to recombinant proteins and complexes.
• To demonstrate the utility of RED-MS for difficult-to-drug targets.

Main Methods:

• Integration of Rapid Equilibrium Dialysis (RED) with Mass Spectrometry (MS) for binding affinity measurements.
• Utilized a single dialysis dataset for affinity calculations.
• Employed compound pooling and automated liquid handling to increase assay throughput.

Main Results:

• RED-MS accurately determined binding affinities, showing strong correlation with Surface Plasmon Resonance (SPR) and Affinity Selection Mass Spectrometry (AS-MS).
• The method proved effective for quantifying small molecule binding to large protein complexes unsuitable for other techniques.
• High throughput analysis of hundreds of measurements per week was achieved.

Conclusions:

• RED-MS provides a reliable solution for measuring compound binding in solution.
• This technique facilitates small molecule affinity optimization, especially for challenging protein targets.
• RED-MS enhances efficiency and throughput in drug discovery research.