Screening of a one bead–one compound combinatorial library for β-actin identifies molecules active toward Ramos B-lymphoma cells

Abstract

The search for small molecules that specifically recognize protein targets is a laborious process if conducted in a one protein-one compound manner. A high-throughput antibody-based screening of one bead-one compound (OBOC) combinatorial small molecule libraries is described here, whereby libraries containing thousands of different small molecule ligands are synthesized on individual TentaGel beads and simultaneously screened for protein binding to individual beads, each with a different compound. The use of OBOC libraries greatly facilitates this simultaneous screening of thousands of compounds. Now, through the use of monoclonal or affinity-purified antibodies, small molecules that bind a particular protein contained in a complex mixture of biological molecules have been identified. This method identified small molecule ligands that bound beta-actin present in cytoplasmic cell extracts of Ramos B-lymphoma cells. These small molecule ligands were resynthesized in immobilized and soluble forms and tested for binding of beta-actin present in Ramos B-cell extracts and for activity against Ramos lymphoma cells. This high-throughput screening immunoassay method has great promise for improving our ability to find relevant, bioactive small molecules that target a specific native protein in a complex protein mixture without purification of the protein.

Screening and identification of mimotopes of the major shrimp allergen tropomyosin using one-bead-one-compound peptide libraries

“…Linear or cyclic mimotopes from six irrelevant allergens ( Supplementary Table 3) were also mapped to Met e 1 using EpiSearch (Table 2, Figure 4d). Only half of the irrelevant inputs matched to Met e 1, and only a prediction based on Pru p 3 overlapped with a short epitope at Met e 1 [25][26][27][28][29][30] ; none overlapped with Pen a 1 epitopes.…”

“…We adopted a modified two-stage subtraction screening approach 25 to screen OBOC libraries (Supplementary Figure 2). For each round of screening, 80 mL of the library (approximately 60 000 beads) was used and the incubation process was carried out in a polypropylene disposable column (Qiagen, Hilden, Germany).…”

The proteome buccaneers: how to unearth your treasure chest via combinatorial peptide ligand libraries

“…It has been demonstrated [23], in fact, that the content of a single bead (potentially carrying a single oligo-peptide structure) after contact with a crude protein extract, is constituted of a few proteins -some dominant and other minor species. In view of discovering specific peptide binders from a large library for the separation of one protein, it has been found that a single bead is capable of capturing more than a single species with presumably different dissociation constants [34,35]. As the interaction phenomenon is governed by the law of mass action, displacements effects are not only dependent on affinity constants but also on the relative concentration of species having affinity for the same peptide ligand.…”

Other Applications of Combinatorial Peptide Libraries

“…This was the case of small ligands active against actin present in Ramos B-lymphoma cells (Miyamoto et al, 2008). The screening procedure was performed by two distinct steps.…”