Method for quantitative protein-ligand affinity measurements in compound mixtures

Area of Science:

• Biochemistry
• Analytical Chemistry
• Drug Discovery

Background:

• Quantitative protein-ligand binding affinity (Kd) measurements are crucial for drug discovery.
• Screening large compound libraries requires efficient and accurate affinity determination methods.
• Existing methods can be limited by the need for purified compounds and precise concentration knowledge.

Purpose of the Study:

• To describe and validate a novel affinity selection-mass spectrometry (AS-MS) method for quantitative Kd measurements.
• To demonstrate the method's utility in large, unpurified combinatorial compound libraries.
• To apply the method for optimizing ligands against the oncology target CDK2.

Main Methods:

• Affinity selection-mass spectrometry (AS-MS) using competitive displacement assays.
• Utilizing internal affinity calibrants for relative affinity ranking.
• AS-MS screening of mixture-based combinatorial libraries, including unpurified compounds.
• Validation using purified compound pools and concentration-insensitive displacement curves.

Main Results:

• The AS-MS method accurately measures relative ligand affinities in mixtures without knowing individual concentrations.
• The technique successfully identified and optimized ligands for the oncology target CDK2.
• Results from unpurified libraries mirrored those from purified compounds, demonstrating robustness.
• Structure-activity relationships were established for identified CDK2 ligands.

Conclusions:

• AS-MS provides a powerful, concentration-insensitive approach for quantitative Kd measurements in large libraries.
• The method facilitates affinity optimization and SAR development using unpurified combinatorial synthesis products.
• AS-MS is a valuable tool for hit triage and lead optimization in drug discovery programs.