Utility of Redundant Combinatorial Libraries in Distinguishing High and Low Quality Screening Hits

Abstract

Large one-bead one-compound (OBOC) combinatorial libraries can be constructed relatively easily by solid-phase split and pool synthesis. The use of resins with hydrophilic surfaces, such as TentaGel, allows the beads to be used directly in screens for compounds that bind selectively to labeled proteins, nucleic acids, or other biomolecules. However, we have found that this method, while useful, has a high false positive rate. In other words, beads that are scored as hits often display compounds that prove to be poor ligands for the target of interest when they are resynthesized and carried through validation trials. This results in a significant waste of time and resources in cases where putative hits cannot be validated without resynthesis. Here, we report that this problem can be largely eliminated through the use of redundant OBOC libraries, where more than one bead displaying the same compound is present in the screen. We show that compounds isolated more than once are likely to be high quality ligands for the target of interest, whereas compounds isolated only once have a much higher likelihood of being poor ligands. While the use of redundant libraries does limit the number of unique compounds that can be screened at one time in this format, the overall savings in time, effort, and materials makes this a more efficient route to the isolation of useful ligands for biomolecules.

A Reversible and Highly Selective Inhibitor of the Proteasomal Ubiquitin Receptor Rpn13 Is Toxic to Multiple Myeloma Cells

“…45 Six molecules (Figure 4) were found more than once in the hit pool (three were found three times and three were found four times) so further efforts were focused on these compounds exclusively. All six of these primary hits were re-synthesized to include a fluorescein for fluorescent polarization (FP) analysis.…”

Recognition of Antigen-Specific B-Cell Receptors from Chronic Lymphocytic Leukemia Patients by Synthetic Antigen Surrogates

“…Work in our laboratory on small molecule-antibody interactions has shown that TentaGel beads are heterogeneous and that a small fraction of the bead population displays the compound at exceedingly high density, which probably allows very weak ligands that happen to be displayed at such densities on these rare beads to stably capture an antibody on the surface due to “kinetic trapping” effects (Doran et al, 2014). …”

Chemical Tools to Monitor and Manipulate the Adaptive Immune System

“…Thankfully, we have recently solved this problem as well. It was shown that these false positives are extremely low affinity antibody ligands that happen to be displayed on a TentaGel bead of unusually high density (the loading of individual beads in a batch of TentaGel beads varies by over 20-fold) (Doran et al, 2014). This likely traps bivalent antibodies kinetically in a microenvironment of high ligand density even though the intrinsic affinity is terrible.…”

“…The idea is that the super-high density beads causing the problem are rare in the population and thus it is highly unlikely that the same ligand found on several different beads is a false positive. Indeed we have found that hits isolated more than once from redundant libraries are almost always of high quality (Doran et al, 2014). This has accelerated our work tremendously.…”