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 over half a 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 develop efficient, high-diversity synthesis protocols for a broad range of chemical scaffolds and benchmark the platform in affinity selections against carbonic anhydrase IX, identifying multiple nanomolar binders. We further apply SELs to flap endonuclease 1 (FEN1) - a disease related DNA-processing enzyme inaccessible to DELs - and discover potent inhibitors. Taken together, screening barcode-free libraries of this scale all at once represents an important development, enables access to novel target classes, and promises substantial impact on both academic and industrial early drug discovery.