Discrete On-DNA Screening: A Frontier for Rapid Hit-to-Lead Optimization Applied to p38α

High-throughput screening (HTS) and DNA-encoded library (DEL) selection are cornerstones of drug discovery but suffer from high operational infrastructure requirements or constraints to affinity-only selections. Herein we report a robust on-DNA compound screening method that bridges this gap, enabling direct functional evaluation of DNA-linked small molecules while bypassing plate-based or selection-wash limitations. To validate this platform, a focused collection of trisubstituted benzamides was synthesized and screened directly on-DNA against p38α MAPK. This methodology rapidly prioritized advanced structures, culminating in the seamless identification of compound I-13. Upon off-DNA synthesis, I-13 was confirmed as a potent p38α inhibitor (IC₅₀ = 65 nM), demonstrating 4-fold selectivity over p38β and excellent selectivity over p38γ and p38δ (>461 fold). While achieving reasonable p38α/β isoform selectivity remains a subject for future optimization, this platform offers a powerful, low-barrier alternative to traditional HTS and DEL for rapid, functional hit-to-lead discovery.