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.

The screening of a DNA‐encoded chemical library against carbonic anhydrase IX (CAIX)‐expressing tumor cells is depicted in the cover image. DNA‐encoding techniques allow the construction and screening of chemical libraries of unprecedented size, but these procedures are typically performed using purified protein targets. While the introduction of cell‐based selections expanded the target scope, selection parameters remained obscure. We report systemic optimizations which translated to the successful discovery of CAIX ligands via cell‐based selections. More information can be found in the Full Paper by J. Scheuermann, D. Neri, et al. (DOI: 10.1002/chem.202100816).