Systematic Evaluation of Washing Conditions to Preserve Low Affinity Binders in DEL Selections

DNA-encoded chemical libraries (DELs) accelerate ligand discovery by combining combinatorial synthesis with parallel affinity selection of DNA-tagged compounds. Conventional DEL selection protocols involve the immobilization of protein targets on a solid support such as magnetic beads. Several washes are performed on the beads after library incubation to remove non-binder library members and to enrich for binding compounds. However, excessive washing could also deplete weak or rapidly dissociating binders. Here, we systematically study the impact of the number of washing steps on DEL hit enrichment during screening of a newly synthesized library named THPP-DEL and based on a central 2-amino-3-(4-(1,2,3,6-tetrahydropyridin-4-yl)phenyl)propanoic acid scaffold and containing ∼8 million compounds. THPP-DEL was screened against Carbonic Anhydrase IX (CAIX), Prostate-Specific Membrane Antigen (PSMA), and Human Serum Albumin (HSA) by systematically varying the number of washing steps from 0 to 20. High-throughput sequencing revealed enriched chemotypes against the three targets, with affinities ranging from low nanomolar to low micromolar concentration values. For all hits, total counts and enrichment factors depended on washing conditions and stabilized after three washing steps. The results presented in this paper define practical selection conditions that preserve micromolar binders, control background levels, and reduce unnecessary hit resynthesis of false positives.