Chemical Diversity of Aspergillus alliaceus Phenotypes: Discovery of brominated bianthrones with activity against triple-negative breast cancer cell lines.

Marine-derived fungi have emerged as a source for novel metabolites with a broad range of bioactivities. However, accessing the full potential of fungi under standard laboratory conditions remains challenging. LC-MS-based metabolomics in combination with varied culture conditions is a fast and powerful tool to detect new metabolites. Here, three developmental forms of the marine-derived fungus Aspergillus alliaceus were analyzed and 14 fungal metabolites, including new brominated polyketides (11-14) were isolated. Structure elucidation relied mainly on 1D and 2D NMR techniques and was supported by low- and high-resolution mass spectrometry and DFT-based computations. We sequenced the A. alliaceus genome, identified the bianthrone-producing biosynthetic gene cluster, and conducted expression analysis on genes involved in sexual development and biosynthesis. The NCI-60 cell line panel revealed selective in vitro activity against triple-negative breast cancer (TNBC) for the halogenated allianthrones and their full anti-proliferative and cytotoxic effects were evaluated in five TNBC cell lines.

Total Synthesis of Chalaniline A: An Aminofulvene Fused Chromone from Vorinostat-Treated Fungus Chalara sp. 6661.

Chalaniline A, an aminofulveno[1,2-b]chromone derivative previously isolated from a vorinostat-treated ascomycete Chalara sp., was prepared in nine steps from orcinol (3,5-dihydroxytoluene). In a key transformation, the tricyclic ring system of the target was generated by a pyrrolidine-catalyzed double annulation between α-(methylsulfinyl)-2,6-dihydroxy-4-methylacetophenone and the ketaldoester, methyl 2,5-dioxopentanoate. The resulting tertiary alcohol (coniochaetone H) was further converted to chalaniline A by operations including dehydration (to yield a hydroxyfulvene), Vilsmeier reaction, and enamine exchange.

Outlining the Hidden Curriculum: Perspectives on Successfully Navigating Scientific Conferences.

Scientific conferences and meetings are valuable opportunities for researchers to network, communicate, and develop knowledge. For early career scientists, conferences can also be intimidating, confusing, and overwhelming, especially without having adequate preparation or experience. In this Perspective, we provide advice based on previous experiences navigating scientific meetings and conferences. These guidelines outline parts of the hidden curriculum around preparing for and attending meetings, navigating conference sessions, networking with other scientists, and participating in social activities while upholding a recommended code of conduct.

Computation-guided scaffold exploration of 2E,6E-1,10-trans/cis-eunicellanes.

Eunicellane diterpenoids are a unique family of natural products containing a foundational 6/10-bicyclic framework and can be divided into two main classes, cis and trans, based on the configurations of their ring fusion at C1 and C10. Previous studies on two bacterial diterpene synthases, Bnd4 and AlbS, revealed that these enzymes form cis- and trans-eunicellane skeletons, respectively. Although the structures of these diterpenes only differed in their configuration at a single position, C1, they displayed distinct chemical and thermal reactivities. Here, we used a combination of quantum chemical calculations and chemical transformations to probe their intrinsic properties, which result in protonation-initiated cyclization, Cope rearrangement, and atropisomerism. Finally, we exploited the reactivity of the trans-eunicellane skeleton to generate a series of 6/6/6 gersemiane-type diterpenes via electrophilic cyclization.

Metabolomics-Guided Discovery, Isolation, Structure Elucidation, and Bioactivity of Myropeptins C-E from Myrothecium inundatum.

The saprotrophic filamentous fungus Myrothecium inundatum represents a chemically underexplored ascomycete with a high number of putative biosynthetic gene clusters in its genome. Here, we present new linear lipopeptides from nongenetic gene activation experiments using nutrient and salt variations. Metabolomics studies revealed four myropeptins, and structural analyses by NMR, HRMS, Marfey's analysis, and ECD assessment for their helical properties established their absolute configuration. A myropeptin biosynthetic gene cluster in the genome was identified. The myropeptins exhibit general nonspecific toxicity against all cancer cell lines in the NCI-60 panel, larval zebrafish with EC50 concentrations of 5-30 µM, and pathogenic bacteria and fungi (MICs of 4-32 µg/mL against multidrug-resistant S. aureus and C. auris). In vitro hemolysis, cell viability, and ionophore assays indicate that the myropeptins target mitochondrial and cellular membranes, inducing cell depolarization and cell death. The toxic activity is modulated by the length of the lipid side chain, which provides valuable insight into their structure-activity relationships.

Total Synthesis of Chalaniline B: An Antibiotic Aminoxanthone from Vorinostat-Treated Fungus Chalara sp. 6661.

Chalaniline B [1-anilino-2,8-dihydroxy-3-(hydroxymethyl)xanthone], an antibiotic previously isolated from vorinostat-treated Chalara sp., was prepared in 7 steps from 2-hydroxyxanthone by a route incorporating regioselective oxidative transformations (bromination at C1/C3, ketone directed Pd(II)-catalyzed hydroxylation at C8), installation of the C1-anilino moiety by a regioselective Buchwald-Hartwig amination reaction from 1,3-dibromo-2,8-dimethoxyxanthone, and late-stage hydroxymethylation at C3 using a Stille cross-coupling. Biological evaluation of deshydroxymethylchalaniline B (1-anilino-2,8-dihydroxyxanthone) revealed MIC values of 8 µg mL-1 (25 µM) against both methicillin resistant S. aureus and B. subtilis.

Biolayer interferometry provides a robust method for detecting DNA binding small molecules in microbial extracts.

DNA replication is an exceptional point of therapeutic intervention for many cancer types and several small molecules targeting DNA have been developed into clinically used antitumor agents. Many of these molecules are naturally occurring metabolites from plants and microorganisms, such as the widely used chemotherapeutic doxorubicin. While natural product sources contain a vast number of DNA binding small molecules, isolating and identifying these molecules is challenging. Typical screening campaigns utilize time-consuming bioactivity-guided fractionation approaches, which use sequential rounds of cell-based assays to guide the isolation of active compounds. In this study, we explore the use of biolayer interferometry (BLI) as a tool for rapidly screening natural product sources for DNA targeting small molecules. We first verified that BLI robustly detected DNA binding using designed GC- and AT-rich DNA oligonucleotides with known DNA intercalating, groove binding, and covalent binding agents including actinomycin D (1), doxorubicin (2), ethidium bromide (3), propidium iodide (4), Hoechst 33342 (5), and netropsin (6). Although binding varied with the properties of the oligonucleotides, measured binding affinities agreed with previously reported values. We next utilized BLI to screen over 100 bacterial extracts from our microbial library for DNA binding activity and found three highly active extracts. Binding-guided isolation was used to isolate the active principle component from each extract, which were identified as echinomycin (8), actinomycin V (9), and chartreusin (10). This biosensor-based DNA binding screen is a novel, low-cost, easy to use, and sensitive approach for medium-throughput screening of complex chemical libraries. Graphical abstract.

Antibacterial Drimane Sesquiterpenes from Aspergillus ustus.

Bioactivity-guided isolation of Aspergillus ustus led to the discovery of five new drimane sesquiterpenes, named ustusal A, ustusolate F and G, and ustusoic acid A and B, 1-5 respectively. Structural elucidation of these fungal terpenes relied on 1D and 2D NMR techniques, high-resolution mass spectrometry, and chiroptical properties. Their relative configurations were determined by NMR methods, while the absolute configurations were established using comparative analyses of computed and experimental NMR chemical shifts and ECD spectra. The sesquiterpenes exhibited weak activity against the clinically relevant pathogens vancomycin-resistant Enterococcus faecium and multidrug-resistant Staphylococcus aureus; however, the activity of 5 was drastically enhanced when equal amounts of stromemycin (6), a known metabolite coisolated from the same fraction from A. ustus, was added.

Biodiversity, Bioactivity, and Metabolites of High Desert Derived Oregonian Soil Bacteria.

From arid, high desert soil samples collected near Bend, Oregon, 19 unique bacteria were isolated. Each strain was identified by 16S rRNA gene sequencing, and their organic extracts were tested for antibacterial and antiproliferative activities. Noteworthy, six extracts (30 %) exhibited strong inhibition resulting in less than 50 % cell proliferation in more than one cancer cell model, tested at 10 µg/mL. Principal component analysis (PCA) of LC/MS data revealed drastic differences in the metabolic profiles found in the organic extracts of these soil bacteria. In total, fourteen potent antibacterial and/or cytotoxic metabolites were isolated via bioactivity-guided fractionation, including two new natural products: a pyrazinone containing tetrapeptide and 7-methoxy-2,3-dimethyl-4H-chromen-4-one, as well as twelve known compounds: furanonaphthoquinone I, bafilomycin C1 and D, FD-594, oligomycin A, chloramphenicol, MY12-62A, rac-sclerone, isosclerone, tunicamycin VII, tunicamycin VIII, and (6S,16S)-anthrabenzoxocinone 1.264-C.

Comparative Genomics of Eight Fusarium graminearum Strains with Contrasting Aggressiveness Reveals an Expanded Open Pangenome and Extended Effector Content Signatures.

Fusarium graminearum, the primary cause of Fusarium head blight (FHB) in small-grain cereals, demonstrates remarkably variable levels of aggressiveness in its host, producing different infection dynamics and contrasted symptom severity. While the secreted proteins, including effectors, are thought to be one of the essential components of aggressiveness, our knowledge of the intra-species genomic diversity of F. graminearum is still limited. In this work, we sequenced eight European F. graminearum strains of contrasting aggressiveness to characterize their respective genome structure, their gene content and to delineate their specificities. By combining the available sequences of 12 other F. graminearum strains, we outlined a reference pangenome that expands the repertoire of the known genes in the reference PH-1 genome by 32%, including nearly 21,000 non-redundant sequences and gathering a common base of 9250 conserved core-genes. More than 1000 genes with high non-synonymous mutation rates may be under diverse selection, especially regarding the trichothecene biosynthesis gene cluster. About 900 secreted protein clusters (SPCs) have been described. Mostly localized in the fast sub-genome of F. graminearum supposed to evolve rapidly to promote adaptation and rapid responses to the host's infection, these SPCs gather a range of putative proteinaceous effectors systematically found in the core secretome, with the chloroplast and the plant nucleus as the main predicted targets in the host cell. This work describes new knowledge on the intra-species diversity in F. graminearum and emphasizes putative determinants of aggressiveness, providing a wealth of new candidate genes potentially involved in the Fusarium head blight disease.

Precursor-Directed Biosynthesis of Aminofulvenes: New Chalanilines from Endophytic Fungus Chalara sp.

The plant endophyte Chalara sp. is able to biotransform the epigenetic modifier vorinostat to form unique, aniline-containing polyketides named chalanilines. Here, we sought to expand the chemical diversity of chalaniline A-type molecules by changing the aniline moiety in the precursor vorinostat. In total, twenty-three different vorinostat analogs were prepared via two-step synthesis, and nineteen were incorporated by the fungus into polyketides. The highest yielding substrates were selected for large-scale precursor-directed biosynthesis and five novel compounds, including two fluorinated chalanilines, were isolated, purified, and structurally characterized. Structure elucidation relied on 1D and 2D NMR techniques and was supported by low- and high-resolution mass spectrometry. All compounds were tested for their bioactivity but were not active in antimicrobial or cell viability assays. Aminofulvene-containing natural products are rare, and this high-yielding, precursor-directed process allows for the diversification of this class of compounds.

Discovery and Biosynthesis of a Structurally Dynamic Antibacterial Diterpenoid.

A new bicyclic diterpenoid, benditerpenoic acid, was isolated from soil-dwelling Streptomyces sp. (CL12-4). We sequenced the bacterial genome, identified the responsible biosynthetic gene cluster, verified the function of the terpene synthase, and heterologously produced the core diterpene. Comparative bioinformatics indicated this Streptomyces strain is phylogenetically unique and possesses nine terpene synthases. The absolute configurations of the new trans-fused bicyclo[8.4.0]tetradecanes were achieved by extensive spectroscopic analyses, including Mosher's analysis, J-based coupling analysis, and computations based on sparse NMR-derived experimental restraints. Interestingly, benditerpenoic acid exists in two distinct ring-flipped bicyclic conformations with a rotational barrier of ≈16 kcal mol-1 in solution. The diterpenes exhibit moderate antibacterial activity against Gram-positive bacteria including methicillin and multi-drug resistant Staphylococcus aureus. This is a rare example of an eunicellane-type diterpenoid from bacteria and the first identification of a diterpene synthase and biosynthetic gene cluster responsible for the construction of the eunicellane scaffold.

Bioenergetics underlying single-cell migration on aligned nanofiber scaffolds.

Cell migration is centrally involved in a myriad of physiological processes, including morphogenesis, wound healing, tissue repair, and metastatic growth. The bioenergetics that underlie migratory behavior are not fully understood, in part because of variations in cell culture media and utilization of experimental cell culture systems that do not model physiological connective extracellular fibrous networks. In this study, we evaluated the bioenergetics of C2C12 myoblast migration and force production on fibronectin-coated nanofiber scaffolds of controlled diameter and alignment, fabricated using a nonelectrospinning spinneret-based tunable engineered parameters (STEP) platform. The contribution of various metabolic pathways to cellular migration was determined using inhibitors of cellular respiration, ATP synthesis, glycolysis, or glucose uptake. Despite immediate effects on oxygen consumption, mitochondrial inhibition only modestly reduced cell migration velocity, whereas inhibitors of glycolysis and cellular glucose uptake led to striking decreases in migration. The migratory metabolic sensitivity was modifiable based on the substrates present in cell culture media. Cells cultured in galactose (instead of glucose) showed substantial migratory sensitivity to mitochondrial inhibition. We used nanonet force microscopy to determine the bioenergetic factors responsible for single-cell force production and observed that neither mitochondrial nor glycolytic inhibition altered single-cell force production. These data suggest that myoblast migration is heavily reliant on glycolysis in cells grown in conventional media. These studies have wide-ranging implications for the causes, consequences, and putative therapeutic treatments aimed at cellular migration.

N-Linked Surface Glycan Biosynthesis, Composition, Inhibition, and Function in Cnidarian-Dinoflagellate Symbiosis.

The success of symbioses between cnidarian hosts (e.g., corals and sea anemones) and micro-algal symbionts hinges on the molecular interactions that govern the establishment and maintenance of intracellular mutualisms. As a fundamental component of innate immunity, glycan-lectin interactions impact the onset of marine endosymbioses, but our understanding of the effects of cell surface glycome composition on symbiosis establishment remains limited. In this study, we examined the canonical N-glycan biosynthesis pathway in the genome of the dinoflagellate symbiont Breviolum minutum (family Symbiodiniaceae) and found it to be conserved with the exception of the transferase GlcNAc-TII (MGAT2). Using coupled liquid chromatography-mass spectrometry (LC-MS/MS), we characterized the cell surface N-glycan content of B. minutum, providing the first insight into the molecular composition of surface glycans in dinoflagellates. We then used the biosynthesis inhibitors kifunensine and swainsonine to alter the glycan composition of B. minutum. Successful high-mannose enrichment via kifunensine treatment resulted in a significant decrease in colonization of the model sea anemone Aiptasia (Exaiptasia pallida) by B. minutum. Hybrid glycan enrichment via swainsonine treatment, however, could not be confirmed and did not impact colonization. We conclude that functional Golgi processing of N-glycans is critical for maintaining appropriate cell surface glycan composition and for ensuring colonization success by B. minutum.

Chemical, Bioactivity, and Biosynthetic Screening of Epiphytic Fungus Zasmidium pseudotsugae.

We report the first secondary metabolite, 8,8'-bijuglone, obtained from pure cultures of the slow growing Douglas fir- (Pseudotsuga menziesii var. menziesii) foliage-associated fungus Zasmidium pseudotsugae. The quinone was characterized using extensive LC/MS and NMR-based spectroscopic methods. 8,8'-Bijuglone exhibited moderate antibiotic activity against Gram-positive pathogens and weak cytotoxic activity in the NCI-60 cell line panel and in our in-house human colon carcinoma (HCT-116) cell line. An analysis of the fungal genome sequence to assess its metabolic potential was implemented using the bioinformatic tool antiSMASH. In total, 36 putative biosynthetic gene clusters were found with a majority encoding for polyketides (17), followed by non-ribosomal peptides (14), terpenes (2), ribosomal peptides (1), and compounds with mixed biosynthetic origin (2). This study demonstrates that foliage associated fungi of conifers produce antimicrobial metabolites and suggests this guild of fungi may present a rich source of novel molecules.

A metabolomics-guided approach to discover Fusarium graminearum metabolites after removal of a repressive histone modification.

Many secondary metabolites are produced by biosynthetic gene clusters (BGCs) that are repressed during standard growth conditions, which complicates the discovery of novel bioactive compounds. In the genus Fusarium, many BGCs reside in chromatin enriched for trimethylated histone 3 lysine 27 (H3K27me3), a modification correlated with transcriptional gene silencing. Here we report on our progress in assigning metabolites to genes by using a strain lacking the H3K27 methyltransferase, Kmt6. To guide isolation efforts, we coupled genetics to multivariate analysis of liquid chromatography-mass spectrometry (LCMS) data from both wild type and kmt6, which allowed identification of compounds previously unknown from F. graminearum. We found low molecular weight, amino acid-derived metabolites (N-ethyl anthranilic acid, N-phenethylacetamide, N-acetyltryptamine). We identified one new compound, protofusarin, as derived from fusarin biosynthesis. Similarly, we isolated large amounts of fusaristatin A, gibepyrone A, and fusarpyrones A and B, simply by using the kmt6 mutant, instead of having to optimize growth media. To increase the abundance of metabolites underrepresented in wild type, we generated kmt6 fus1 double mutants and discovered tricinolone and tricinolonoic acid, two new sesquiterpenes belonging to the tricindiol class. Our approach allows rapid visualization and analyses of the genetically induced changes in metabolite production, and discovery of new molecules by a combination of chemical and genetic dereplication. Of 22 fungal metabolites identified here, 10 compounds had not been reported from F. graminearum before. We show that activating silent metabolic pathways by mutation of a repressive chromatin modification enzyme can result in the discovery of new chemistry even in a well-studied organism, and helps to connect new or known small molecules to the BGCs responsible for their production.

Expression of a Structural Protein of the Mycovirus FgV-ch9 Negatively Affects the Transcript Level of a Novel Symptom Alleviation Factor and Causes Virus Infection-Like Symptoms in Fusarium graminearum.

Infections of fungi by mycoviruses are often symptomless but sometimes also fatal, as they perturb sporulation, growth, and, if applicable, virulence of the fungal host. Hypovirulence-inducing mycoviruses, therefore, represent a powerful means to defeat fungal epidemics on crop plants. Infection with Fusarium graminearum virus China 9 (FgV-ch9), a double-stranded RNA (dsRNA) chrysovirus-like mycovirus, debilitates Fusarium graminearum, the causal agent of fusarium head blight. In search for potential symptom alleviation or aggravation factors in F. graminearum, we consecutively infected a custom-made F. graminearum mutant collection with FgV-ch9 and found a mutant with constantly elevated expression of a gene coding for a putative mRNA-binding protein that did not show any disease symptoms despite harboring large amounts of virus. Deletion of this gene, named virus response 1 (vr1), resulted in phenotypes identical to those observed in the virus-infected wild type with respect to growth, reproduction, and virulence. Similarly, the viral structural protein coded on segment 3 (P3) caused virus infection-like symptoms when expressed in the wild type but not in the vr1 overexpression mutant. Gene expression analysis revealed a drastic downregulation of vr1 in the presence of virus and in mutants expressing P3. We conclude that symptom development and severity correlate with gene expression levels of vr1 This was confirmed by comparative transcriptome analysis, showing a large transcriptional overlap between the virus-infected wild type, the vr1 deletion mutant, and the P3-expressing mutant. Hence, vr1 represents a fundamental host factor for the expression of virus-related symptoms and helps us understand the underlying mechanism of hypovirulence.IMPORTANCE Virus infections of phytopathogenic fungi occasionally impair growth, reproduction, and virulence, a phenomenon referred to as hypovirulence. Hypovirulence-inducing mycoviruses, therefore, represent a powerful means to defeat fungal epidemics on crop plants. However, the poor understanding of the molecular basis of hypovirulence induction limits their application. Using the devastating fungal pathogen on cereal crops, Fusarium graminearum, we identified an mRNA binding protein (named virus response 1, vr1) which is involved in symptom expression. Downregulation of vr1 in the virus-infected fungus and vr1 deletion evoke virus infection-like symptoms, while constitutive expression overrules the cytopathic effects of the virus infection. Intriguingly, the presence of a specific viral structural protein is sufficient to trigger the fungal response, i.e., vr1 downregulation, and symptom development similar to virus infection. The advancements in understanding fungal infection and response may aid biological pest control approaches using mycoviruses or viral proteins to prevent future Fusarium epidemics.

Biological evaluation of molecules of the azaBINOL class as antiviral agents: Inhibition of HIV-1 RNase H activity by 7-isopropoxy-8-(naphth-1-yl)quinoline.

Inspired by bioactive biaryl-containing natural products found in plants and the marine environment, a series of synthetic compounds belonging to the azaBINOL chiral ligand family was evaluated for antiviral activity against HIV-1. Testing of 39 unique azaBINOLs and two BINOLs in a single-round infectivity assay resulted in the identification of three promising antiviral compounds, including 7-isopropoxy-8-(naphth-1-yl)quinoline (azaBINOL B#24), which exhibited low-micromolar activity without associated cytotoxicity. The active compounds and several close structural analogues were further tested against three different HIV-1 envelope pseudotyped viruses as well as in a full-virus replication system (EASY-HIT). The in vitro studies indicated that azaBINOL B#24 acts on early stages of viral replication before viral assembly and budding. Next we explored B#24's activity against HIV-1 reverse transcriptase (RT) and individually tested for polymerase and RNase H activity. The azaBINOL B#24 inhibits RNase H activity and binds directly to the HIV-1 RT enzyme. Additionally, we observe additive inhibitory activity against pseudotyped viruses when B#24 is dosed in competition with the clinically used non-nucleoside reverse transcriptase inhibitor (NNRTI) efavirenz. When tested against a multi-drug resistant HIV-1 isolate with drug resistance associated mutations in regions encoding for HIV-1 RT and protease, B#24 only exhibits a 5.1-fold net decrease in IC50 value, while efavirenz' activity decreases by 7.6-fold. These results indicate that azaBINOL B#24 is a potentially viable, novel lead for the development of new HIV-1 RNase H inhibitors. Furthermore, this study demonstrates that the survey of libraries of synthetic compounds, designed purely with the goal of facilitating chemical synthesis in mind, may yield unexpected and selective drug leads for the development of new antiviral agents.

Polyketides from Marine-Derived Aspergillus porosus: Challenges and Opportunities for Determining Absolute Configuration.

Fungal natural products have inspired and enabled countless modern therapeutics. During a survey of the secondary metabolites of endophytic fungi, we found that Aspergillus porosus produces new polyketides with interesting structural features named porosuphenols A-D (1, 2, 3a, and 3b). The structural elucidation of these metabolites was performed with 1D and 2D NMR techniques, Mosher ester analysis, J-based conformational analysis, and isotope exchange studies. The absolute configuration of these compounds was determined using typical approaches including comparative analysis of experimental NMR and electronic circular dichroism spectra with DFT calculations. However, these efforts did not provide conclusive results for porosuphenol A (1). To resolve this issue, we applied a strategy in which NMR data guide the conformer search. Herein are presented the structure elucidation of porosuphenols A-D as a case study in the challenges and opportunities for determination of absolute configuration. Lastly, bioassay-guided fractionation of cytotoxic fractions resulted in the additional isolation of pimarane diterpenes, sphaeropsidin A (4), and aspergiloid E (5).

The natural product mensacarcin induces mitochondrial toxicity and apoptosis in melanoma cells.

Mensacarcin is a highly oxygenated polyketide that was first isolated from soil-dwelling Streptomyces bacteria. It exhibits potent cytostatic properties (mean of 50% growth inhibition = 0.2 µm) in almost all cell lines of the National Cancer Institute (NCI)-60 cell line screen and relatively selective cytotoxicity against melanoma cells. Moreover, its low COMPARE correlations with known standard antitumor agents indicate a unique mechanism of action. Effective therapies for managing melanoma are limited, so we sought to investigate mensacarcin's unique cytostatic and cytotoxic effects and its mode of action. By assessing morphological and biochemical features, we demonstrated that mensacarcin activates caspase-3/7-dependent apoptotic pathways and induces cell death in melanoma cells. Upon mensacarcin exposure, SK-Mel-28 and SK-Mel-5 melanoma cells, which have the BRAFV600E mutation associated with drug resistance, showed characteristic chromatin condensation as well as distinct poly(ADP-ribose)polymerase-1 cleavage. Flow cytometry identified a large population of apoptotic melanoma cells, and single-cell electrophoresis indicated that mensacarcin causes genetic instability, a hallmark of early apoptosis. To visualize mensacarcin's subcellular localization, we synthesized a fluorescent mensacarcin probe that retained activity. The natural product probe was localized to mitochondria within 20 min of treatment. Live-cell bioenergetic flux analysis confirmed that mensacarcin disturbs energy production and mitochondrial function rapidly. The subcellular localization of the fluorescently labeled mensacarcin together with its unusual metabolic effects in melanoma cells provide evidence that mensacarcin targets mitochondria. Mensacarcin's unique mode of action suggests that it may be a useful probe for examining energy metabolism, particularly in BRAF-mutant melanoma, and represent a promising lead for the development of new anticancer drugs.