Natural Product Discovery Efforts Leading to the Isolation of Spliceosome-Modulating Oxazolomycins

Abstract

Natural products remain a cornerstone of therapeutic discovery; yet many known metabolites were discovered before modern multi-omics strategies. This dissertation illustrates a combinatorial chemical biology framework integrating biosynthetic gene cluster knowledge and elicitation, multiplexed phenotypic screening, and integrated omics to uncover previously unrecognized biological activities from established natural product scaffolds. Utilization of the Multiplexed Activity Mapping (MAM) platform enabled functional screening of 70 cave-derived actinomycetes for metabolites inducing distinct cell death phenotypic responses in acute myeloid leukemia (AML). This revealed the macrolide narbomycin induces DNA damage alongside apoptotic and autophagic responses while DNA damage is largely uninvestigated for the class. Additionally, the anthracycline isoquinocycline B elicited cell-cycle arrest and DNA damage profiles distinct from doxorubicin, highlighting functional divergence within clinically used scaffolds. Building on these rediscovery efforts, a pharmacophore-guided genome mining campaign targeting the spirocyclic β-lactone (SβL) moiety found in oxazolomycins enabled the identification of oxazolomycin D family metabolites from an unreported producer strain, Streptomyces platensis. Multiplexed single-cell phenotypic profiling revealed suppression of phospho-S6 phosphorylation corresponding to these observed metabolomic features, prompting further investigation. Isolation of four known and one novel oxazolomycin D family members enabled cytotoxicity profiling and flow-cytometric analyses suggesting that the oxazolomycins 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. This structural framework motivated focused investigation of early signaling consequences, revealing rapid suppression of phospho-S6 and perturbation of nutrient-sensing pathways. Global cellular thermal proteome profiling analysis of oxazolomycin D 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 oxazolomycin D. The combinatorial approaches presented in this body of work illustrates multiple applications leading to novel biological phenotypes to known and discarded natural product scaffolds.