Spirocyclic β-lactone secondary metabolites modulate spliceosome function

, which contained a cryptic SβL-producing gene cluster. Utilizing single cell Multiplexed Activity Metabolomics to assess a panel of regulated cell injury and cell death markers in metabolomic arrays tested against MV-4-11 cells, phosphoprotein-S6 phosphorylation inhibition was observed for several metabolomic features, prompting us to prioritize these metabolites for structure elucidation. Several compounds were isolated including one new variant, permitting correlation of a cryptic biosynthetic gene cluster entry for oxazolomycin D (OxD) family metabolites. Cytotoxicity and flow cytometric structure-activity relationships for isolated analogs were determined using an expanded phospho-flow metabolism marker panel suggesting that SβLs possess two pharmacophores: a mixed polyketide component, involved in target engagement and inhibition, and a nonribosomal peptide synthetase-derived covalent β-lactone warhead, contributing to cytotoxicity. Global cellular thermal proteome profiling analysis of OxD treated cells implicated potent interaction with and modulation of spliceosome associated proteins. Spliceosome-associated functional changes were validated by alternate splicing analysis upon induction by OxD. As aberrant RNA splicing occurs in nearly all cancer types, the spliceosome-associated cellular response profiles of OxD define an unexplored opportunity for therapeutic development in cancers dependent upon alternative splicing.