AS-TLC-Based High-Throughput Screening Integrated with CuAAC Optimization Strategy Facilitates the Rapid Discovery of...

The classical sequential workflow-individual compound screening, followed by stepwise design-synthesis-purification of derivatives-makes lead compound discovery time-consuming and cost-prohibitive. Herein, we developed Affinity Selection Thin-Layer Chromatography (AS-TLC) as a method for high-throughput screening (HTS), enabling efficient and cost-effective screening of compound mixtures. Furthermore, we integrated activity assays with AS-TLC to minimize target protein consumption and lower the false positive rate, through which we identified a YTH domain-containing protein 1 (YTHDC1) inhibitor, fragment YD, with a half-maximal inhibitory concentration (IC₅₀) of 20.01 ± 3.30 µm. Building on this hit, we applied copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry for rapid structural modification and compound library construction, ultimately obtaining LC-YD03 with significantly enhanced inhibitory activity (IC₅₀ = 41.98 ± 6.24 nm). The interaction mode between LC-YD03 and YTHDC1 was further clarified via X-ray crystallographic analysis. This study demonstrates that the AS-TLC-activity assay platform enables efficient hit screening from compound mixtures; subsequent click chemistry-based combinatorial library technology accelerates lead compound optimization. Collectively, this workflow significantly reduces the time and cost associated with lead compound discovery.