Barcode-free hit discovery from massive libraries enabled by automated small molecule structure annotation

Affinity-selection platforms are powerful tools in early drug discovery, but current technologies – most notably DNA-encoded libraries (DELs) – are limited by synthesis complexity and incompatibility with nucleic acid-binding targets. We present a barcode-free self-encoded library (SEL) platform that enables direct screening of ~1 million small molecules in a single experiment. SELs combine tandem mass spectrometry with custom software for automated structure annotation, eliminating the need for external tags for the identification of screening hits. We developed efficient, high-diversity synthesis protocols for a broad range of chemical scaffolds and benchmarked the platform in affinity selections against carbonic anhydrase IX, identifying multiple nanomolar binders. We further applied SELs to flap endonuclease 1 (FEN1) – a disease related DNA-processing enzyme inaccessible to DELs – and discovered potent inhibitors. Taken together, screening barcode-free libraries of this scale is unprecedented, enables access to novel target classes, and promises substantial impact on both academic and industrial early drug discovery.