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

Abstract

<jats:p> 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 chromodomain of CBX8. The results indicate low DNA tag bias and show that affinity-based in vitro selection assays are sufficiently robust for both ligand discovery and determination of quantitative structure–activity relationships. </jats:p>

Quantitative Assessment of Affinity Selection Performance by Using DNA‐Encoded Chemical Libraries

“…Our observations are largely in keeping with the results of Krusemark and co‐workers, who studied the efficiency of affinity capture selections by using DNA‐encoded peptides, which were capable of binding to CBX . The authors found that lower enrichments and recoveries of ligands were obtained for lower ligand affinity to both CBX7 and CBX8 ChDs .…”

“…Our observations are largely in keeping with the results of Krusemark and co‐workers, who studied the efficiency of affinity capture selections by using DNA‐encoded peptides, which were capable of binding to CBX . The authors found that lower enrichments and recoveries of ligands were obtained for lower ligand affinity to both CBX7 and CBX8 ChDs . If the findings of both studies are confirmed for other proteins, there might be a need to develop more efficient selection methods, particularly for targets for which only hits with micromolar potency can be expected.…”

Design and Construction of a Focused DNA-Encoded Library for Multivalent Chromatin Reader Proteins

“…Targeting chromatin reader proteins using DELs requires an approach to diminish non-specific DNA interactions interfering during the selections, as many of these proteins are highly basic and in fact coordinate with DNA [68][69][70]. Studies from the Krusemark lab have shown the utility of DELs for chromodomain containing proteins by producing a DEL of peptidomimetics [71,72]. To address the feasibility of DELs for chromatin readers in our hands and to establish general selection and sequencing protocols, we explored a model system using the chromodomain-containing Kme reader protein CBX7, which is part of the multi-subunit protein complex Polycomb Repressive Complex 1 [56].…”

Encoded Library Technologies as Integrated Lead Finding Platforms for Drug Discovery

“…This may also contribute to the observation that a larger number of DEL literature reports describe the identification of enzyme inhibitors binding to well defined pockets [8]. Nonetheless, the number of examples successfully identifying binders to challenging targets like PPIs [63,65,66,88,89] and MoAs such as agonists, positive allosteric modulators and antagonists for G-protein coupled receptors (GPCRs) [67,68,90,91,92] grows at an increasing rate. Key contributors to the recent success against GPCRs were advances in production of membrane proteins [93] and stabilization of specific conformations [94,95].…”