Actinomycetota bioprospecting from ore-forming environments.

Natural products from Actinomycetota have served as inspiration for many clinically relevant therapeutics. Despite early triumphs in natural product discovery, the rate of unearthing new compounds has decreased, necessitating inventive approaches. One promising strategy is to explore environments where survival is challenging. These harsh environments are hypothesized to lead to bacteria developing chemical adaptations (e.g. natural products) to enable their survival. This investigation focuses on ore-forming environments, particularly fluoride mines, which typically have extreme pH, salinity and nutrient scarcity. Herein, we have utilized metagenomics, metabolomics and evolutionary genome mining to dissect the biodiversity and metabolism in these harsh environments. This work has unveiled the promising biosynthetic potential of these bacteria and has demonstrated their ability to produce bioactive secondary metabolites. This research constitutes a pioneering endeavour in bioprospection within fluoride mining regions, providing insights into uncharted microbial ecosystems and their previously unexplored natural products.

A Diels-Alder probe for discovery of natural products containing furan moieties.

Natural products (NPs) are fantastic sources of inspiration for novel pharmaceuticals, oftentimes showing unique bioactivity against interesting targets. Specifically, NPs containing furan moieties show activity against a variety of diseases including fungal infections, and cancers. However, it is challenging to discover and isolate these small molecules from cell supernatant. The work described herein showcases the development of a molecular probe that can covalently modify furan moieties via a [4 + 2] Diels-Alder cycloaddition, making them easily identifiable on liquid chromatography-mass spectrometry (LC-MS). The molecular probe, which undergoes this reaction with a variety of furans, was designed with both a UV-tag and a mass tag to enable easy identification. The probe has been tested with a variety of purified furans, including natural products, methylenomycin furan (MMF) hormones, and MMF derivatives. Moreover, the molecular probe has been tested in crude supernatants of various Streptomyces strains and enables identification of MMFs.

Discovery of Streptomyces species CS-62, a novel producer of the Acinetobacter baumannii selective antibiotic factumycin.

Narrow-spectrum antibiotics are of great interest given their ability to spare the microbiome and decrease widespread antibiotic resistance compared to broad-spectrum antibiotics. Herein, we screened an in-house library of Actinobacteria strains for selective activity against Acinetobacter baumannii and successfully identified Streptomyces sp. CS-62 as a producer of a natural product with this valuable activity. Analysis of the cultures via high-resolution mass spectrometry and tandem mass spectrometry, followed by comparison with molecules in the Natural Product Atlas and the Global Natural Products Social Molecular Networking platform, suggested a novel natural product. Genome mining analysis initially supported the production of a novel kirromycin derivative. Isolation and structure elucidation via mass spectrometry and Nuclear Magnetic Resonance (NMR) analyses revealed that the active natural product was the known natural product factumycin, exposing omissions and errors in the consulted databases. While public databases are generally very useful for avoiding rediscovery of known molecules, rediscovery remains a problem due to public databases either being incomplete or having errors that result in failed dereplication. Overall, the work describes the ongoing problem of dereplication and the continued need for public database curation.

Small molecule inducers of actinobacteria natural product biosynthesis.

Actinobacteria are a large and diverse group of bacteria that are known to produce a wide range of secondary metabolites, many of which have important biological activities, including antibiotics, anti-cancer agents, and immunosuppressants. The biosynthesis of these compounds is often highly regulated with many natural products (NPs) being produced at very low levels in laboratory settings. Environmental factors, such as small molecule elicitors, can induce the production of secondary metabolites. Specifically, they can increase titers of known NPs as well as enabling discovery of novel NPs typically produced at undetectable levels. These elicitors can be NPs, including antibiotics or hormones, or synthetic compounds. In recent years, there has been a growing interest in the use of small molecule elicitors to induce the production of secondary metabolites from actinobacteria, especially for the discovery of NPs from "silent" biosynthetic gene clusters. This review aims to highlight classes of molecules that induce secondary metabolite production in actinobacteria and to describe the potential mechanisms of induction. ONE-SENTENCE SUMMARY: This review describes chemical elicitors of actinobacteria natural products described to date and the proposed mechanisms of induction.

Stapled Peptides as Direct Inhibitors of Nrf2-sMAF Transcription Factors.

Nuclear factor erythroid-related 2-factor 2 (Nrf2) is a transcription factor traditionally thought of as a cellular protector. However, in many cancers, Nrf2 is constitutively activated and correlated with therapeutic resistance. Nrf2 heterodimerizes with small musculoaponeurotic fibrosarcoma Maf (sMAF) transcription factors, allowing binding to the antioxidant responsive element (ARE) and induction of transcription of Nrf2 target genes. While transcription factors are historically challenging to target, stapled peptides have shown great promise for inhibiting these protein-protein interactions. Herein, we describe the first direct cell-permeable inhibitor of Nrf2/sMAF heterodimerization. N1S is a stapled peptide designed based on AlphaFold predictions of the interactions between Nrf2 and sMAF MafG. A cell-based reporter assay combined with in vitro biophysical assays demonstrates that N1S directly inhibits Nrf2/MafG heterodimerization. N1S treatment decreases the transcription of Nrf2-dependent genes and sensitizes Nrf2-dependent cancer cells to cisplatin. Overall, N1S is a promising lead for the sensitization of Nrf2-addicted cancers.

An Unusual Oxidative Rearrangement Catalyzed by a Divergent Member of the 2-Oxoglutarate-Dependent Dioxygenase Superfamily during Biosynthesis of Dehydrofosmidomycin.

The biosynthesis of the natural product dehydrofosmidomycin involves an unusual transformation in which 2-(trimethylamino)ethylphosphonate is rearranged, desaturated and demethylated by the enzyme DfmD, a divergent member of the 2-oxoglutarate-dependent dioxygenase superfamily. Although other members of this enzyme family catalyze superficially similar transformations, the combination of all three reactions in a single enzyme has not previously been observed. By characterizing the products of in vitro reactions with labeled and unlabeled substrates, we show that DfmD performs this transformation in two steps, with the first involving desaturation of the substrate to form 2-(trimethylamino)vinylphosphonate, and the second involving rearrangement and demethylation to form methyldehydrofosmidomycin. These data reveal significant differences from the desaturation and rearrangement reactions catalyzed by other family members.

One-shot access to α,ß-difunctionalized azepenes and dehydropiperidines by reductive cross-coupling of α-selenonyl-ß-selenyl enamides with organic bromides.

The synthesis of α- and α,ß-functionalized azepenes and dehydropiperidines from readily prepared α-selenonyl eneformamides or enecarbamates has been achieved through Fe-catalyzed α-substitutive deselenonation, ß-regioselective lithiation/trapping, and Co-catalyzed reductive cross-coupling protocols.

Trapping of carbolithiation-derived tertiary benzylic α-lithio piperidines with carbon electrophiles: Controlling the formation of α-amino quaternary and vicinal stereocenters.

The interception of carbolithiation-derived tertiary benzylic α-lithio piperidines with carbon electrophiles, under HMPA-mediated conditions, has led to the diastereoselective synthesis of vicinally functionalized piperidines bearing α-amino quaternary stereocenters.

Direct access to functionalized benzotropones, azepanes, and piperidines by reductive cross-coupling of α-bromo enones with α-bromo enamides.

The synthesis of functionalized azepenes and piperidines bearing an α-cycloheptenone or benzotropone derivative has been accomplished through direct reductive cross-coupling of α-bromo eneformamides or enecarbamates with highly versatile α-bromo benzotropone derivatives, under cobalt catalysis. The coupling products have been further elaborated to other synthetically useful aza-heterocyclic frameworks.

Expedient access to α,ß-difunctionalized azepenes using α-halo eneformamides: application to the one-pot synthesis of 2-benzazepanes.

The regioselective synthesis of α,ß-difunctionalized (alkenyl, aryl, sulfonyl, allyl, or alkynyl) azepenes has been accomplished through α-halo eneformamides. A successful implementation of the vicinal functionalization strategy has led to a one-pot synthesis of 2-benzazepanes whose benzenoid portion is highly functionalized.