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.
- SourceAvailable from: Andrew J S Knox[show abstract] [hide abstract]
ABSTRACT: In eukaryotic cells, proteins and RNAs are transported between the nucleus and the cytoplasm by nuclear import and export receptors. Over the past decade, small molecules that inhibit the nuclear export receptor CRM1 have been identified, most notably leptomycin B. However, up to now no small molecule inhibitors of nuclear import have been described. Here we have used our automated confocal nanoscanning and bead picking method (CONA) for on-bead screening of a one-bead one-compound library to identify the first such import inhibitor, karyostatin 1A. Karyostatin 1A binds importin β with high nanomolar affinity and specifically inhibits importin α/β mediated nuclear import at low micromolar concentrations in vitro and in living cells, without perturbing transportin mediated nuclear import or CRM1 mediated nuclear export. Surface plasmon resonance binding experiments suggest that karyostatin 1A acts by disrupting the interaction between importin β and the GTPase Ran. As a selective inhibitor of the importin α/β import pathway, karyostatin 1A will provide a valuable tool for future studies of nucleocytoplasmic trafficking.ACS Chemical Biology 10/2010; 5(10):967-79. · 5.44 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: An enzyme labeling and screening strategy for the discovery of ligands selective in binding two structurally similar members of the aldo-keto reductase family of enzymes is reported. The resulting fluorescence microscope data obtained by screening a 74,088 member library led to the identification of selective ligands for aldose reductase (ALR2) and aldehyde reductase (ALR1). Resynthesis results validate the selectivity of these ligands.Bioorganic & Medicinal Chemistry Letters 07/2005; 15(11):2938-42. · 2.34 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Proteolysis of short N alpha-protected peptide substrates bound to polyoxyethylene-polystyrene beads releases selectively free amino sites in the enzyme-accessible "surface" area. The substantial majority of functional sites in the "interior" of the polymeric support are not reached by the enzyme and remain uncleaved (protected). Subsequent synthesis with two classes of orthogonal protecting groups-N alpha-tert-butyloxycarbonyl (Boc) and N alpha-9-fluorenylmethyloxy-carbonyl (Fmoc)-allows generation of two structures on the same bead. The surface structure is available for receptor interactions, whereas the corresponding interior structure is used for coding. Coding structures are usually readily sequenceable peptides. This "shaving" methodology was illustrated by the preparation of a peptide-encoded model peptide combinatorial library containing 1.0 x 10(5) members at approximately 6-fold degeneracy. From this single library, good ligands were selected for three different receptors: anti-beta-endorphin anti-body, streptavidin, and thrombin, and the binding structures were deduced correctly by sequencing the coding peptides present on the same beads.Proceedings of the National Academy of Sciences 09/1996; 93(16):8194-9. · 9.74 Impact Factor