Affinity Selections of DNA‐Encoded Chemical Libraries on Carbonic Anhydrase IX‐Expressing Tumor Cells Reveal a Dependence on Ligand Valence

Abstract

DNA-encoded chemical libraries are typically screened against purified protein targets. Recently, cell-based selections with encoded chemical libraries have been described, commonly revealing suboptimal performance due to insufficient recovery of binding molecules. We used carbonic anhydrase IX (CAIX)-expressing tumor cells as a model system to optimize selection procedures with code-specific quantitative polymerase chain reaction (qPCR) as selection readout. Salt concentration and performing PCR on cell suspension had the biggest impact on selection performance, leading to 15-fold enrichment factors for high-affinity monovalent CAIX binders (acetazolamide; K D = 8.7 nM). Surprisingly, the homobivalent display of acetazolamide at the extremities of both complementary DNA strands led to a substantial improvement of both ligand recovery and enrichment factors (above 100-fold). The optimized procedures were used for selections with a DNA-encoded chemical library comprising 1 million members against tumor cell lines expressing CAIX, leading to a preferential recovery of known and new ligands against this validated tumor-associated target. This work may facilitate future affinity selections on cells against target proteins which might be difficult to express otherwise.

Converting Double-Stranded DNA-Encoded Libraries (DELs) to Single-Stranded Libraries for More Versatile Selections

“… 88 , 89 Neri and co-workers also reported cell-based selections against CA-9, another membrane carbonic anhydrase isoform using dual-pharmacophore DELs. 90 CBS is a known ligand for CA-12 ( K d = 0.97 μM) 88 and can be used as a positive control in the selection. 88 , 89 We first tested whether the CBS-conjugated ssDNA, obtained from λ exo digestion, could selectively capture the CA-12 protein on live cells.…”

Recent advances in DNA-encoded dynamic libraries

“…2a). 32,37,[97][98][99][100][101][102] The library is selected against immobilized targets, and the binding BBs can be decoded by sequencing the coding regions. Initially, the two BBs in a binding pair are separately decoded and their correlation is lost; thus, ESAC libraries were mostly used for affinity maturation of known ligands.…”

Evolution of the Selection Methods of DNA-Encoded Chemical Libraries

“…The authors did not estimate the target concentration, but the cell density was high (2 × 10 6 cells/50 μL); therefore, a high effective molarity of DOR is expected. Recently, Neri and co-workers comprehensively optimized the experimental parameters for cell-based DEL selections . Interestingly, they observed that bivalent ligands significantly improved the compound enrichment, suggesting that multivalency effect may be another way to drive the binding equilibrium on the cell surface.…”

“…Recently, Neri and co-workers comprehensively optimized the experimental parameters for cell-based DEL selections. 72 Interestingly, they observed that bivalent ligands significantly improved the compound enrichment, suggesting that multivalency effect may be another way to drive the binding equilibrium on the cell surface. However, this remains to be further examined with large-scale DEL selections and more targets.…”