Spirocyclic β-lactone secondary metabolites modulate spliceosome function

Abstract

Spirocyclic β-lactones (SβLs) are ring-strain activated natural products produced by actinomycetota, possessing potent and selective cytotoxicity, and with an unknown pharmacological mechanism. To further the understanding of their mechanism of action and the discovery of new SβLs, we obtained a series of known and previously not isolated SβLs from Streptomyces platensis , 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.