Affinity ligand selection from a library of small molecules: assay development, screening, and application

Area of Science:

• Biochemistry
• Chemical Biology
• Drug Discovery

Background:

• Developing efficient methods for identifying high-affinity ligands is crucial for drug discovery and proteomics.
• Traditional screening methods can be time-consuming, costly, and prone to high false-positive rates, especially when screening compound mixtures.

Purpose of the Study:

• To describe a facile, cost-effective, high-throughput 96-well plate filtration method for screening synthetic libraries for affinity ligands.
• To demonstrate the method's efficacy using human serum albumin (HSA) as a target protein and assess its performance with both discrete compounds and mixtures.

Main Methods:

• A 96-well plate filtration assay was developed for screening compounds immobilized on a solid support.
• Fluorescamine-based fluorescence detection was used for quantifying bound target protein (HSA) in the filtrate.
• The method was validated by testing 100,000 compounds (25,000 discrete, 75,000 in mixtures) and performing secondary screening for specificity and binding capacity.

Main Results:

• An overall hit rate of 8% was observed across all tested compounds.
• Screening mixtures with high protein binding led to a high hit rate upon deconvolution, unlike previous methods prone to false positives.
• A single-step purification of HSA from serum using an affinity column derived from a lead compound achieved purity comparable to two-step ion-exchange chromatography.

Conclusions:

• The developed high-throughput filtration method is a simple, inexpensive, and effective tool for primary screening of large compound libraries.
• The method accurately identifies affinity ligands from both discrete samples and complex mixtures, significantly reducing false positives.
• This approach is broadly applicable to any soluble target protein and holds potential as a generic chemical affinity tool for proteomics research.