biostudies-literatureVinogradov AAJapan Agency for Medical Research and DevelopmentJapan Society for the Promotion of Science20332-20341https://www.ebi.ac.uk/biostudies/studies/S-EPMC9650704biostudies-literatureUnknown144(44)Bioengineering of ribosomally synthesized and post-translationally modified peptides (RiPPs) is an emerging approach to explore the diversity of pseudo-natural product structures for drug discovery purposes. However, despite the initial advances in this area, bioactivity reprogramming of multienzyme RiPP biosynthetic pathways remains a major challenge. Here, we report a platform for de novo discovery of functional thiopeptides based on reengineered biosynthesis of lactazole A, a RiPP natural product assembled by five biosynthetic enzymes. The platform combines in vitro biosynthesis of lactazole-like thiopeptides and mRNA display to prepare and screen large (≥10¹²) combinatorial libraries of pseudo-natural products. We demonstrate the utility of the developed protocols in an affiJournal of the American Chemical SocietyDe Novo Discovery of Thiopeptide Pseudo-natural Products Acting as Potent and Selective TNIK Kinase Inhibitors.PMC9650704JP21H04712JP16H06444JP19K22243JP20H05618JP20K15407JP19am0101070JP20H02866Hamada KNishimura HTerasaka NZhang YGoto YSengoku TSuga HVinogradov AAOnaka HOkada COgata KChang JSfalseDe Novo Discovery of Thiopeptide Pseudo-natural Products Acting as Potent and Selective TNIK Kinase Inhibitors.Bioengineering of ribosomally synthesized and post-translationally modified peptides (RiPPs) is an emerging approach to explore the diversity of pseudo-natural product structures for drug discovery purposes. However, despite the initial advances in this area, bioactivity reprogramming of multienzyme RiPP biosynthetic pathways remains a major challenge. Here, we report a platform for de novo discovery of functional thiopeptides based on reengineered biosynthesis of lactazole A, a RiPP natural product assembled by five biosynthetic enzymes. The platform combines in vitro biosynthesis of lactazole-like thiopeptides and mRNA display to prepare and screen large (≥10¹²) combinatorial libraries of pseudo-natural products. We demonstrate the utility of the developed protocols in an affi2022-01-01T00:00:00Z2022 Nov2026-05-31T23:07:47.76Z2025-04-04T21:00:48.047ZS-EPMC96507043628292210.1021/jacs.2c07937