AS‐TLC‐Based High‐Throughput Screening Integrated with CuAAC Optimization Strategy Facilitates the Rapid Discovery of YTHDC1 Inhibitor

ABSTRACT 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 (IC 50 ) of 20.01 ± 3.30 µ m . 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 (IC 50 = 41.98 ± 6.24 n m ). 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.