Discovery, Structural Studies and Biosynthesis of Biologically Active Natural Products and Natural Product-Like Inhibitors

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

The past year has yielded significant advances in natural product discovery, biosynthetic enzymology, and structural biology, with particular emphasis on Mass Spectrometry (MS) methods aimed at identifying understudied molecular scaffolds, novel RiPPs, bioactive polyketides, and the mechanisms of action of new antibacterials. One major achievement in 2025 is the discovery and biosynthesis of the aridomycins—a new class of tetramic acid antifungal, antibiotics. This work pushed the limits of spectroscopic characterization of complex molecules, and employed extensive NMR analyses, J-based configurational analyses, utilization of Kishi’s Universal NMR Databases, to assign the absolute configuration of 28 stereocenters in a complex polyhydroxylated polyketide natural product. Whole genome sequencing and bioinformatics of the two Amycolatopsis spp. producing strains permitted validation of the absolute configurations and identified a previously unrecognized acyltransferase specificity motif (GHSQ···FVAH) associated with hydroxymalonyl-ACP incorporation. Using temporal biosynthetic studies, we also demonstrated that aridomycin is synthesized in situ as a glucosylated compound that is devoid of antibacterial activity. Within the biosynthetic gene cluster, a dedicated glycosyl transferase and glycosidase produce the pro-drug and active compound, respectively, indicating that the producing organism uses a pro-drug strategy for self-resistance or protection. In other major efforts we developed an approach that combines MS data-derived molecular networking with MS database-derived mass defect analysis to preferentially discover new compounds with high structural novelty in the initial stage of a discovery workflow. Specifically, we showed that unknown metabolites or clusters generated from molecular networking can be assigned to a compound class based on their relative mass defects (RMDs) calculated using open-source databases. When combined with a single type of ancillary data such as ultraviolet or MS/MS spectra, the method goes a step further and can identify unknown clusters, or compound types, as new scaffolds when the data are incongruent with their RMD class. We validated the approach through the discovery of the brasiliencins, a new macrolide family bearing glycosylation. Biological activity studies showed the brasiliencins to be potent antimycobacterials, with activity dependent on a single chiral center. Additional natural product discovery efforts focused on identifying antibacterial compounds with activity against multidrug-resistant (MDR) pathogens, including MRSA and VRE. Several newly isolated secondary metabolites showed potent inhibition of bacterial growth in vitro and in cell-based assays. Mechanistic studies revealed diverse modes of action, including direct targeting of phospholipids (work in preparation). Looking forward, we aim to expand our structural biology efforts toward new classes of RiPP enzymes, develop structure-guided mutagenesis campaigns to tailor bioactivity, and leverage new genomics and metabolomics platforms to mine underexplored environments for novel biosynthetic gene clusters.