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

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 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.

Keywords: DNA-encoded libraries; affinity selection assay; chromobox (CBX) proteins; chromodomains; peptidomimetics.

Figure 1

Optimized selection scheme with His₆-tagged CBX7 and CBX8 using three on-DNA peptide ligands and nonligand.

Figure 2

(A) On-DNA PSL parental peptide (PP) sequence. Colored amino acids indicate varied amino acids at the P(–1), P(–2), P(–3), and P(–4) positions. The invariant amino acids (Ser and Kme₃) are shown in black. (B) Parallel synthesis of on-DNA PSL members.

Figure 3

Dot blot of enrichments of DNA-encoded PSL with selection against either CBX8 or CBX7 at the lowest (~2.5 μM) protein concentration. The color of each dot represents the log enrichment relative to the nonligand control included in each selection.

Figure 4

(A) Comparison of PSL members for selectivity toward CBX8 or CBX7. (B) Summary of SAR observed from on-DNA PSL selections against CBX7 and CBX8.