Barcode-free hit discovery from massive libraries enabled by automated small molecule structure annotation
- Edith van der Nol
- Nils Alexander Haupt
- Qing Qing Gao
- Benthe A. M. Smit
- Martin Andre Hoffmann
- Martin Engler-Lukajewski
- Marcus Ludwig
- Sean McKenna
- J. Miguel Mata
- Olivier J. M. Béquignon
- Gerard van Westen
- Tiemen J. Wendel
- Sylvie M. Noordermeer
- Sebastian Böcker
- Sebastian Pomplun
2025-10-27
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.