On-bead screens sample narrower affinity ranges of protein-ligand interactions compared to equivalent solution assays

Abstract

Conceptually, on-bead screening is one of the most efficient high-throughput screening (HTS) methods. One of its inherent advantages is that the solid support has a dual function: it serves as a synthesis platform and as a screening compartment. Compound purification, cleavage and storage and extensive liquid handling are not necessary in bead-based HTS. Since the establishment of one-bead one-compound library synthesis, the properties of polymer beads in chemical reactions have been thoroughly investigated. However, the characterization of the kinetics and thermodynamics of protein-ligand interactions on the beads used for screening has received much less attention. Consequently, the majority of reported on-bead screens are based on empirically derived procedures, independent of measured equilibrium constants and rate constants of protein binding to ligands on beads. More often than not, on-bead screens reveal apparent high affinity binders through strong protein complexation on the matrix of the solid support. After decoding, resynthesis, and solution testing the primary hits turn out to be unexpectedly weak binders, or may even fall out of the detection limit of the solution assay. Only a quantitative comparison of on-bead binding and solution binding events will allow systematically investigating affinity differences as function of protein and small molecule properties. This will open up routes for optimized bead materials, blocking conditions and other improved assay procedures. By making use of the unique features of our previously introduced confocal nanoscanning (CONA) method, we investigated the kinetic and thermodynamic properties of protein-ligand interactions on TentaGel beads, a popular solid support for on-bead screening. The data obtained from these experiments allowed us to determine dissociation constants for the interaction of bead-immobilized ligands with soluble proteins. Our results therefore provide, for the first time, a comparison of on-bead versus solution binding thermodynamics. Our data indicate that affinity ranges found in on-bead screening are indeed narrower compared to equivalent interactions in homogeneous solution. A thorough physico-chemical understanding of the molecular recognition between proteins and surface bound ligands will further strengthen the role of on-bead screening as an ultimately cost-effective method in hit and lead finding.