Moroidins are plant ribosomally synthesized and posttranslationally modified peptides (RiPPs) called burpitides biosynthesized from copper-dependent peptide cyclases. The bicyclic structure of moroidins contains (1) a stephanotic acid scaffold with a Leu-Cꞵ-Trp-indole-C6-crosslink and (2) a C-terminal ring formed by a Trp-indole-C2-His-imidazole-N1-crosslink. Moroidin is cytotoxic to H1437 non-small cell lung adenocarcinoma cells in vitro, underscoring the potential of stephanotic acid-type burpitides as anticancer lead structures and the importance of exploring diversification strategies to discover analogs with enhanced bioactivity. We mined the transcriptome of 7579 plant species from 498 plant families to identify moroidin analogs with novel second-ring structures and the cyclases responsible for their biosynthesis. A search of >27000 candidate burpitide cyclases reveals two stephanotic acid-type burpitides in plants with new second-ring crosslinks derived from posttranslational modification: Glechomanin from ground ivy (Glechoma hederacea) with a C-C-crosslink between a C-terminal tryptophan-indole-C6 and the β-carbon of a valine, and Mercurialin from annual mercury (Mercurialis annua) featuring a C-O-crosslink between a C-terminal tyrosine-phenol hydroxy and the β-carbon of a phenylalanine, respectively. Furthermore, our transcriptomics-guided burpitide genotyping enabled isolation of a moroidin analog from water chickweed (Stellaria aquatica), which exhibits a ten-fold higher in vitro cytotoxicity than moroidin and selective toxicity against H1437 lung adenocarcinoma cells. We demonstrate that plant transcriptome mining can expand the medicinal chemistry toolbox for chemical and biological exploration of burpitide lead structures.
