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Open Access Publications from the University of California

Scholarly Works (10 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2019)

By the turn of the 21th century, it became evident that even microorganisms intensely studied as sources of natural products contained the DNA instructions to produce many more compounds than those that were being extracted. Thus, in order to tap into this source of novel natural products, which could serve as structural leads for the development of new drugs against emerging diseases like cancer and multi-resistant bacterial infections, new methods are urgently needed. In this work, a method dubbed “reactivity-guided isolation” is investigated as a drug discovery approach for different classes of natural products: 1) electrophilic natural products, a therapeutically-relevant class of natural products that covalently modifies their cellular targets, 2) conjugated alkene-containing natural products, a characteristic moiety found in natural products of polyketide origin, and 3) isocyanide-containing natural products, a functional group associated with the biological activity observed in various sponge metabolites. Using carefully designed chemoselective reagents, the method forms derivatives of these different classes of natural products that are UV-active and highly conspicuous via mass spectrometry (MS) by virtue of an isotopically-unique bromine or chlorine tag. Throughout development and application of this method we have expanded the toolbox for derivatizing natural products (and the reagents) and in the process we have synthesized high-value derivatives from high yielding coupling reactions; we have gain insight into the use of UV and MS tags and crystallization units for X-ray analysis that provide opportunities to simplify detection and structural elucidation of natural products; and we have discovered new natural products entities from microorganisms grown in the laboratory, and in some cases predict how they might interact with cellular components. The work presented here has the potential to streamline natural product discovery platforms and lead to new compounds useful in the treatment of diseases.

    Cover page: Reactivity-guided isolation of marine and bacterial natural products in the genomic age
    • Thesis
    • Peer Reviewed
    UC San Diego Electronic Theses and Dissertations (2019)

    Marine natural products have long served as leads for pharmaceutical compounds, as well as sources of compounds whose value lies in the novelty of their structures. As more natural products are discovered and described, compounds with truly novel structures or bioactivities have become increasingly difficult to find. Therefore, a reactivity-guided approach has been developed to aid in the search for natural products bearing specific functional groups. An azide-based probe for terminal alkyne-bearing natural products was synthesized and implemented, which facilitated the discovery of the vatiamide family of natural products. Further, a suite of tetrazine-based probes for isonitrile-bearing natural products was synthesized and evaluated on the basis of reactivity, stability, and fragmentation behavior in mass spectrometry.

    The marine bacterial natural product lymphostin was recognizable for its unusual structural features, but its biochemical activity, and mechanism thereof, was not fully understood. Lymphostin and its family of related analogs were evaluated for their kinase inhibition activity, and model systems were synthesized to understand lymphostin’s mechanism of kinase inhibition. Lymphostin was found to be a potent and irreversible inhibitor of PI3K/mTOR, the first compound from bacteria to be shown with this activity. An effort towards the total synthesis of lymphostin was then undertaken, resulting in the discovery of an unusual heteroaryl dimerization reaction.

      Cover page: Reactivity-Guided Isolation and Medicinal Chemistry of Marine Bacterial Natural Products
      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2017)
      Members of the actinomycete genus Streptomyces are non-motile, filamentous bacteria that are well-known for the production of biomedically relevant secondary metabolites. While considered obligate aerobes, little is known about how these bacteria respond to periods of reduced oxygen availability in their natural habitats, which include soils and ocean sediments. Here, we provide evidence that the marine streptomycete strain CNQ-525 can reduce MnO2 via a diffusible mechanism. We investigated the effects of hypoxia on secondary metabolite production and observed a shift away from the antibiotic napyradiomycin towards 8-amino-flaviolin, an intermediate in the napyradiomycin biosynthetic pathway. We purified 8-amino-flaviolin and demonstrated that it is reversibly redox-active (midpoint potential -474.5 mV), indicating that it has the potential to function as an endogenous extracellular electron shuttle. This study provides evidence that environmentally triggered changes in secondary metabolite production may provide clues to the ecological functions of specific compounds, and that Gram-positive bacteria considered to be obligate aerobes may play previously unrecognized roles in biogeochemical cycling through mechanisms that include extracellular electron shuttling.
        Cover page: Ecological implications of hypoxia‐triggered shifts in secondary metabolism
        • Article
        • Peer Reviewed
        UC San Diego Previously Published Works (2018)
        Bacterial genome sequences consistently contain many more biosynthetic gene clusters encoding specialized metabolites than predicted by the compounds discovered from the respective strains. One hypothesis invoked to explain the cryptic nature of these gene clusters is that standard laboratory conditions do not provide the environmental cues needed to trigger gene expression. A potential source of such cues is other members of the bacterial community, which are logical targets for competitive interactions. In this study, we examined the effects of such interactions on specialized metabolism in the marine actinomycete Salinispora tropica. The results show that antibiotic activities and the concentration of some small molecules increase in the presence of co-occurring bacterial strains relative to monocultures. Some increases in antibiotic activity could be linked to nutrient depletion by the competitor as opposed to the production of a chemical cue. Other increases were correlated with the production of specific compounds by S. tropica. In particular, one interaction with a Vibrio sp. consistently induced antibiotic activity and was associated with parent ions that were unique to this interaction, although the associated compound could not be identified. This study provides insight into the metabolomic complexities of bacterial interactions and baseline information for future genome mining efforts.
          Cover page: The role of inter-species interactions in Salinispora specialized metabolism
          • Article
          • Peer Reviewed
          UC San Diego Previously Published Works (2019)
          Hybrid type I PKS/NRPS biosynthetic pathways typically proceed in a collinear manner wherein one molecular building block is enzymatically incorporated in a sequence that corresponds to gene arrangement. In this work, genome mining combined with the use of a fluorogenic azide-based click probe led to the discovery and characterization of vatiamides A-F, three structurally diverse alkynylated lipopeptides, and their brominated analogues, from the cyanobacterium Moorea producens ASI16Jul14-2. These derive from a unique combinatorial non-collinear PKS/NRPS system encoded by a 90 kb gene cluster in which an upstream PKS cassette interacts with three separate cognate NRPS partners. This is facilitated by a series of promiscuous intermodule PKS-NRPS docking motifs possessing identical amino acid sequences. This interaction confers a new type of combinatorial capacity for creating molecular diversity in microbial systems.
            Cover page: Nature's Combinatorial Biosynthesis Produces Vatiamides A–F
            • Article
            • Peer Reviewed
            UC San Diego Previously Published Works (2022)
            The obligate marine actinobacterial genus Salinispora has become a model organism for natural product discovery, yet little is known about the ecological functions of the compounds produced by this taxon. The aims of this study were to assess the effects of live cultures and culture extracts from two Salinispora species on invertebrate predators. In choice-based feeding experiments using the bacterivorous nematode Caenorhabditis elegans, live cultures of both Salinispora species were less preferred than Escherichia coli. When given a choice between the two species, C. elegans preferred S. areniolca over S. tropica. Culture extracts from S. tropica deterred C. elegans, while those from S. arenicola did not, suggesting that compounds produced by S. tropica account for the feeding deterrence. Bioactivity-guided isolation linked compounds in the lomaiviticin series to the deterrent activity. Additional assays using the marine polychaete Ophryotrocha siberti and marine nematodes further support the deterrent activity of S. tropica against potential predators. These results provide evidence that Salinispora natural products function as a defense against predation and that the strategies of predation defense differ between closely related species. IMPORTANCE Bacteria inhabiting marine sediments are subject to predation by bacterivorous eukaryotes. Here, we test the hypothesis that sediment-derived bacteria in the genus Salinispora produce biologically active natural products that function as a defense against predation. The results reveal that cultures and culture extracts of S. tropica deter feeding by Caenorhabditis elegans and negatively affect the habitat preference of a marine annelid (Ophryotrocha siberti). These activities were linked to the lomaiviticins, a series of cytotoxic compounds produced by S. tropica. Microbial natural products that function as a defense against predation represent a poorly understood trait that can influence community structure in marine sediments.
              Cover page: Specialized Metabolite-Mediated Predation Defense in the Marine Actinobacterium Salinispora
              • Article
              • Peer Reviewed
              UC San Diego Previously Published Works (2018)
              Using a novel chemistry-based assay for identifying electrophilic natural products in unprocessed extracts, we identified the PI3-kinase/mTOR dual inhibitor neolymphostin A from Salinispora arenicola CNY-486. The method further showed that the vinylogous ester substituent on the neolymphostin core was the exact site for enzyme conjugation. Tandem MS/MS experiments on PI3Kα treated with the inhibitor revealed that neolymphostin covalently modified Lys802 with a shift in mass of +306 amu, corresponding to addition of the inhibitor and elimination of methanol. The binding pose of the inhibitor bound to PI3Kα was modeled, and hydrogen-deuterium exchange mass spectrometry experiments supported this model. Against a panel of kinases, neolymphostin showed good selectivity for PI3-kinase and mTOR. In addition, the natural product blocked AKT phosphorylation in live cells with an IC50 of ∼3 nM. Taken together, neolymphostin is the first reported example of a covalent kinase inhibitor from the bacterial domain of life.
                Cover page: Neolymphostin A Is a Covalent Phosphoinositide 3‑Kinase (PI3K)/Mammalian Target of Rapamycin (mTOR) Dual Inhibitor That Employs an Unusual Electrophilic Vinylogous Ester
                • Article
                • Peer Reviewed
                UC San Diego Previously Published Works (2022)
                Fungal infection of grasses, including rice (Oryza sativa), sorghum (Sorghum bicolor), and barley (Hordeum vulgare), induces the formation and accumulation of flavonoid phytoalexins. In maize (Zea mays), however, investigators have emphasized benzoxazinoid and terpenoid phytoalexins, and comparatively little is known about flavonoid induction in response to pathogens. Here, we examined fungus-elicited flavonoid metabolism in maize and identified key biosynthetic enzymes involved in the formation of O-methylflavonoids. The predominant end products were identified as two tautomers of a 2-hydroxynaringenin-derived compound termed xilonenin, which significantly inhibited the growth of two maize pathogens, Fusarium graminearum and Fusarium verticillioides. Among the biosynthetic enzymes identified were two O-methyltransferases (OMTs), flavonoid OMT 2 (FOMT2), and FOMT4, which demonstrated distinct regiospecificity on a broad spectrum of flavonoid classes. In addition, a cytochrome P450 monooxygenase (CYP) in the CYP93G subfamily was found to serve as a flavanone 2-hydroxylase providing the substrate for FOMT2-catalyzed formation of xilonenin. In summary, maize produces a diverse blend of O-methylflavonoids with antifungal activity upon attack by a broad range of fungi.
                  Cover page: Biosynthesis and antifungal activity of fungus-induced O-methylated flavonoids in maize
                  • Article
                  • Peer Reviewed
                  UC San Diego Previously Published Works (2022)
                  The identity and biological activity of most metabolites still remain unknown. A bottleneck in the exploration of metabolite structures and pharmaceutical activities is the compound purification needed for bioactivity assignments and downstream structure elucidation. To enable bioactivity-focused compound identification from complex mixtures, we develop a scalable native metabolomics approach that integrates non-targeted liquid chromatography tandem mass spectrometry and detection of protein binding via native mass spectrometry. A native metabolomics screen for protease inhibitors from an environmental cyanobacteria community reveals 30 chymotrypsin-binding cyclodepsipeptides. Guided by the native metabolomics results, we select and purify five of these compounds for full structure elucidation via tandem mass spectrometry, chemical derivatization, and nuclear magnetic resonance spectroscopy as well as evaluation of their biological activities. These results identify rivulariapeptolides as a family of serine protease inhibitors with nanomolar potency, highlighting native metabolomics as a promising approach for drug discovery, chemical ecology, and chemical biology studies.
                    Cover page: Native metabolomics identifies the rivulariapeptolide family of protease inhibitors
                    • Article
                    • Peer Reviewed
                    UC Davis Previously Published Works (2018)
                    Natural products and their derivatives continue to be wellsprings of nascent therapeutic potential. However, many laboratories have limited resources for biological evaluation, leaving their previously isolated or synthesized compounds largely or completely untested. To address this issue, the Canvass library of natural products was assembled, in collaboration with academic and industry researchers, for quantitative high-throughput screening (qHTS) across a diverse set of cell-based and biochemical assays. Characterization of the library in terms of physicochemical properties, structural diversity, and similarity to compounds in publicly available libraries indicates that the Canvass library contains many structural elements in common with approved drugs. The assay data generated were analyzed using a variety of quality control metrics, and the resultant assay profiles were explored using statistical methods, such as clustering and compound promiscuity analyses. Individual compounds were then sorted by structural class and activity profiles. Differential behavior based on these classifications, as well as noteworthy activities, are outlined herein. One such highlight is the activity of (-)-2(S)-cathafoline, which was found to stabilize calcium levels in the endoplasmic reticulum. The workflow described here illustrates a pilot effort to broadly survey the biological potential of natural products by utilizing the power of automation and high-throughput screening.
                      Cover page: Canvass: A Crowd-Sourced, Natural-Product Screening Library for Exploring Biological Space.Creative Commons 'BY' version 4.0 license
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