Robustness of In Vitro Selection Assays of DNA-Encoded Peptidomimetic Ligands to CBX7 and CBX8

SLAS Discovery
Kyle E DentonCasey J Krusemark

Abstract

The identification of protein ligands from a DNA-encoded library is commonly conducted by an affinity selection assay. These assays are often not validated for robustness, raising questions about selections that fail to identify ligands and the utility of enrichment values for ranking ligand potencies. Here, we report a method for optimizing and utilizing affinity selection assays to identify potent and selective peptidic ligands to the highly related chromodomains of CBX proteins. To optimize affinity selection parameters, statistical analyses (Z' factors) were used to define the ability of selection assay conditions to identify and differentiate ligands of varying affinity. A DNA-encoded positional scanning library of peptidomimetics was constructed around a trimethyllysine-containing parent peptide, and parallel selections against the chromodomains from CBX8 and CBX7 were conducted over three protein concentrations. Relative potencies of off-DNA hit molecules were determined through a fluorescence polarization assay and were consistent with enrichments observed by DNA sequencing of the affinity selection assays. In addition, novel peptide-based ligands were discovered with increased potency and selectivity to the chromodomai...Continue Reading

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Citations

Feb 27, 2020·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Justin M RectenwaldKenneth H Pearce
May 22, 2018·SLAS Discovery·Robert Goodnow
Aug 2, 2019·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Dongwook KimCasey J Krusemark
Jul 17, 2020·Frontiers in Molecular Biosciences·Joseph M Rogers
Dec 10, 2020·ChemMedChem·Verena B K KunigAndreas Brunschweiger
Feb 13, 2019·Journal of the American Chemical Society·James P PhelanGary A Molander
Jun 27, 2021·ChemMedChem·Natalia MilosevichFraser Hof

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Methods Mentioned

BETA
pull-down
PCR
phage display
affinity
affinity selection
PCA
electrophoresis
PCRs
Assay
dot-blot

Software Mentioned

GraphPad Prism

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