Seamless Bead to Microarray Screening: Rapid Identification of the Highest Affinity Protein Ligands from Large Combinatorial Libraries

Abstract

Summary Several approaches have been developed for screening combinatorial libraries or collections of synthetic molecules for agonists or antagonists of protein function, each with its own advantages and limitations. In this report, we describe an experimental platform that seamlessly couples massively parallel bead-based screening of one bead one compound combinatorial libraries with microarray-based quantitative comparisons of the binding affinities of the many hits isolated from the bead library. Combined with other technical improvements, this technique allows the rapid identification of the best protein ligands in combinatorial libraries containing millions of compounds without the need for labor-intensive re-synthesis of the hits.

Potential pharmacological chaperones targeting cancer-associated MCL-1 and Parkinson disease-associated α-synuclein

“…For the rapid and efficient selection of best compounds among a library of α-helix mimetics, we developed a convenient HTS method by taking advantage of a bead-based screening platform, which was originally developed by Lam and colleagues for screening peptides (42) and has been successfully applied to cyclic peptides and peptidomimetics such as β-peptides and peptoids (29,(43)(44)(45)(46)(47). Relative to conventional HTS assays, on-bead screening has several advantages (48).…”

“…For selection of the best MCL-1 ligands from the library molecules, we executed two rounds of on-bead screens, using magnetic separation followed by alkaline phosphatase-based detection (SI Appendix) (44,47). Briefly, 25 mg (∼10,200 beads, about seven copies of diversity) of the library beads were screened for their binding ability to MCL-1ΔNΔC (amino acids 172-320), a BH3-binding domain of MCL-1, by incubating the library beads with 200 nM biotinylated MCL-1ΔNΔC overnight at 4°C.…”

Other Applications of Combinatorial Peptide Libraries

“…To facilitate the separation of positive beads from the bead bulk, a specific investigative work has been made by Astle et al (2010) with an interesting proposal. After incubation with the protein mixture containing the target protein, the bulk beads are incubated with the antibody against the Schematic representation of multiple protein docking on the same bead (same peptide sequence) with true affinity and nonspecific binding.…”

Combinatorial/Library Peptide Synthesis

“…Astle et al [271] simplified the process of hit isolation through magnetic bead sorting. The target protein was labeled with a magnetic nanoparticle.…”

“…The structure of selected hit is then deduced by tandem MS. In this way, no resynthesis of the hits is necessary until the best binders are confirmed [271]. (Illustration adapted from [272].)…”