Massive barcode-free chemical screenings enable the discovery of bioactive macrocycles with passive membrane permeability.

Synthetic macrocycles offer exceptional potential as therapeutics. However, most high-throughput discovery platforms rely on genetically encoded libraries of large peptide macrocycles, which typically are not optimized for drug like properties. Fully synthetic libraries offer greater flexibility in accessing broader chemical space. Leveraging recent advances in mass spectrometry based library techniques, here we report CycloSEL (Cyclic Self-Encoded Libraries), an end-to-end workflow, that screens synthetic macrocycle libraries enriched in drug-like 'beyond rule of five' features. The workflow relies on affinity selections and hit identification by tandem mass spectrometry, eliminating the need for genetic barcodes. We construct a 16 million-member library and validate the approach against the oncology target carbonic anhydrase IX, achieving robust enrichment and accurate identification of true binders. Applying CycloSEL to the acute myeloid leukemia target WD repeat-containing protein 5 (WDR5) yields a macrocycle with subnamolar affinity, and potent inhibition of the WDR5-Mixed-Lineage Leukemia 1 (MLL1) interaction. Subsequent modifications produce a chameleonic macrocycle with passive membrane permeability, serum stability, and anti-proliferative activity in leukemia cells. Together, these results demonstrate that CycloSEL enables discovery of drug-like macrocycles from fully synthetic libraries for intracellular targets.