The growing threat of multidrug-resistant bacterial infections highlights the urgent need for antibiotics with novel mechanisms of action. Gromomycins, a newly identified class of triterpene antibiotics, exhibit potent activity against Gram-positive bacteria, including drug-resistant species, through a previously uncharacterized mode of action. Here, we report the discovery of a gromomycin-like biosynthetic gene cluster in the Actinoplanes genus through a genome mining approach, leading to the isolation and characterization of new bioactive derivatives that overcome resistance to clinically used drugs in vancomycin-resistant enterococci. Mechanistic studies revealed that gromomycins induce rapid potassium ion leakage and depolarization of the bacterial membrane, resulting in bactericidal activity against Staphylococcus aureus. Gromomycins disrupt the integrity of the cytoplasmic membrane, as evidenced by large pore formation, leakage of intracellular contents, and subsequent cell lysis. Supplementation with membrane lipids and fatty acids neutralized their antibacterial activity, suggesting a direct membrane-targeting mechanism, further supported by the inability to raise gromomycin resistance and their toxic effects on eukaryotic cells. Collectively, these findings deepen our understanding of gromomycin activity and demonstrate the utility of genome mining to uncover structurally novel and biologically active natural products.

Hepatic fructose utilization depends on ketohexokinase mediated phosphorylation to generate fructose-1-phosphate and commit fructose carbons to additional metabolic steps. Since dysregulated fructose metabolism has been directly connected to the onset and progression of liver disease and cancer, there is considerable interest in identifying the contributions of fructose carbons in bioenergetic pathways. An essential technology for assessing fructose utilization has been the application of isotopically labeled fructose and magnetic resonance with the development of 13C hyperpolarized imaging with [2-13C]fructose allowing for in vivo assessments. While hyperpolarized imaging of [2-13C]fructose has achieved remarkable success in the detection of cancer metabolism, this approach has yet to be utilized to assess fed and fasted states in healthy livers. By challenging mice with a 6 h fast, we demonstrate that hyperpolarized [U-2H, 2-13C]fructose in vivo spectroscopy can clearly distinguish direct hepatic gluconeogenesis. Comprehensively, this work aims to establish a foundational methodology for the assessment of hepatic metabolism in vivo.

Precise regulation of transcriptional dynamics underlies gene expression programs, governing critical biological processes such as cell fate determination, tissue development, and stress responses. While nascent RNA sequencing technologies offer powerful tools for dissecting these mechanisms, existing methods remain constrained by complex workflows, high cellular input requirements, and cytotoxicity. Here, we present Li-BrU-seq, a systematically optimized 5-bromouridine (BrU)-based profiling strategy designed for low-input samples. Rigorous benchmarking demonstrates that Li-BrU-seq outperforms previous protocols in both enrichment specificity and sensitivity. By streamlining the enrichment workflow, the method enables high-quality transcriptomic profiling from low-input material (500 ng total RNA or ∼25,000 cells). Furthermore, Li-BrU-seq supports flexible temporal resolution ranging from ultrashort pulses to long-term tracking, free from the stress-induced artifacts inherent to 4sU. Additionally, it offers tailored workflows compatible with diverse downstream applications. Li-BrU-seq provides an accessible and versatile platform that expands nascent RNA analysis to low-input, rare, and physiologically sensitive biological systems.

Bacterial membrane vesicles (MVs) are natural delivery systems for biomolecules, such as enzymes and nucleic acids, but their role in transporting specialized metabolites is less understood. Many microbial metabolites are lipophilic and poorly water-soluble, raising questions about how they perform ecological functions in aquatic environments. Here, we demonstrate that Pseudoalteromonas piscicida JC3, a marine bacterium with probiotic potential, packages lipophilic depsipeptides known as bromoalterochromides (BACs) into outer membrane vesicles. Untargeted metabolomics and molecular networking identified six known and two previously unknown BACs, while targeted LC-MS/MS localized BACs to MVs and cells, with no detection in culture supernatants. Structure elucidation of a new analogue, bromoalterochromide E/E', was achieved through isolation and spectroscopic analysis, including modified Marfey's analysis to determine amino acid composition and chirality. Functional assays showed that BAC-loaded MVs exhibit antibacterial activity against Staphylococcus aureus and the marine pathogen Vibrio anguillarum, linking vesicle-mediated metabolite delivery to microbial competition. These findings highlight MVs as transporters of lipophilic natural products and suggest their potential as natural drug delivery vehicles in clinical and aquaculture settings.

Poly-ADP-ribosylation (PARylation) is a reversible post-translational modification that occurs in higher eukaryotes. While thousands of PARylated substrates have been identified, the specific biological functions of most PARylated proteins remain elusive. PARylation stoichiometry is a critical parameter to assess the potential functions of a PARylated protein. Here, we developed a large-scale strategy to measure the stoichiometries of protein PARylation. By integrating chemically mild cell lysis conditions, boronate enrichment, and carefully designed titration experiments, we were able to determine the PARylation stoichiometries for a total of 235 proteins. Importantly, this approach enables the capture of all PARylation events, regardless of their amino acid acceptor linkages. We revealed that PARylation occupancy spans over 3 orders of magnitude. However, most PARylation events occur at low stoichiometric values (median 0.58%). Notably, we observed that high-stoichiometry PARylation (>1%) predominantly targets proteins involved in transcription regulation and chromatin remodeling. Thus, our study provides a system-scale, quantitative view of PARylation stoichiometries under genotoxic conditions, which serves as an invaluable resource for future functional studies of this important protein post-translational modification.

The oncogene MYCN is predominantly expressed in cancer stem-like cells, where it drives tumor growth, metastasis, and therapeutic resistance in hepatocellular carcinoma (HCC). In this study, we explored MYCN Inhibitors (MI) from the RIKEN Natural Products Depository chemical library and identified NPD15261 (designated as MI102) as a selective small-molecule inhibitor of MYCN expression. MI102 markedly reduced MYCN mRNA and protein levels in HCC cells, suppressing proliferation and colony formation, while inducing apoptosis, with minimal impact on normal hepatic cells. Mechanistically, kinase profiling revealed that MI102 is a highly selective inhibitor of MET receptor tyrosine kinase that specifically blocks phosphorylation at Y1234/Y1235. Hepatocyte growth factor-mediated MET activation induces MYCN expression and partially rescues MI102-mediated MYCN suppression. Notably, MI102 effect exhibited superior tumor cell selectivity compared with the MET inhibitor tivantinib. At the transcriptional level, RNA-seq revealed that MI102 globally downregulated MYCN-associated oncogenic programs. Collectively, these findings establish pharmacological downregulation of MYCN as a promising therapeutic strategy for HCC and reveal a functional link between MET signaling and MYCN-driven oncogenic pathways.

Actinobacteria are a rich source of bioactive compounds and unique biosynthetic chemistry. Micromonospora echinospora subsp. challisensis NRRL 12255 produces the aromatic polyketide TLN-05220, which exhibits nanomolar activity against antibiotic-resistant human pathogens, including vancomycin-resistant Enterococcus faecalis and methicillin-resistant Staphylococcus aureus. The pentangular polyphenol core of TLN-05220 is decorated with a piperazinone moiety; yet, the enzymes responsible for the construction of this uncommon modification from amino acid precursors are unknown. Synthetic piperazinone-containing molecules have diverse antimicrobial, antiviral, anticancer, and anti-inflammatory bioactivity profiles, and determining biosynthetic routes for the assembly of this heterocycle may enhance drug discovery and medicinal chemistry efforts. We identified a putative TLN-05220 biosynthetic gene cluster (BGC) in the commercially available strain M. echinospora ATCC 15837 that contains both type-I and type-II polyketide synthases, two predicted asparagine synthetase-like enzymes, and two genes (tln1 and tln5) that putatively encode pyridoxal 5'-phosphate (PLP)-dependent amino acid synthases. Stable isotopic feeding studies coupled with liquid chromatography-mass spectrometry (LC-MS) identified l-alanine, l-serine, and glycine as metabolic precursors of TLN-05220. Subsequent in vitro enzymology established that Tln1 is a PLP-dependent alanine racemase, while Tln5 performs a stereoselective β-substitution reaction of O-phospho-l-serine with a preferential d-alanine nucleophile. Alanine racemization and pseudodipeptide l-serine-Cβ-N-d-alanine (d,l-PDP) incorporation into TLN-05220 were further supported using deuterated intermediates and mass spectrometry techniques. Establishing the enzymes that catalyze amino acid installation within TLN-05220 inspires further biosynthetic discovery and engineering while enabling the biocatalytic syntheses of novel amino acid-containing polyketide antibiotics.

Quantitative live cell monitoring of catalytic activity is essential for advancing chemical biology, yet designing substrate probes that combine broad applicability with finely tunable kinetics remains a significant challenge. While glyco-bisacetal-based substrates (BABS) have proven applicable to several enzymes, their alkyl-hemiacetal core can limit turnover rates for certain enzymes. Herein, we report a novel one-pot, three-component glycosylation strategy to synthesize Aryl-BABS through the trapping of transient aryl-hemiacetals. This approach enables rapid diversification of the bisacetal scaffold using various phenols, yielding a library of aryl-bisacetal substrates. Kinetic evaluation of catalytic hydrolysis with a model glycosidase demonstrated that these Aryl-BABS are efficiently processed, with turnover rates up to 2 orders of magnitude faster than analogous alkyl glycosides and approaching those seen for activated p-nitrophenyl glycosides. Simple substitutions to phenol lead to a 20-fold range of kinetic tunability. Crucially, stopped-flow studies combined with kinetic simulations revealed that the breakdown of the enzymatically released aryl-hemiacetal is extremely rapid, at least 100-fold faster than that of alkyl-hemiacetals. This synthetic and kinetic tunability offers a powerful roadmap for developing advanced substrate probes of biocatalysts, eventually enabling quantitative measurement of previously intractable enzymes in living systems.

Mitochondria are believed to be a potential drug target in cancer therapies because of their critical and multiple biofunctions in supplying energy and regulating signaling pathways for cell cycle and proliferation. It has been known that mitochondrial DNA (mtDNA) contains many guanine-rich sequences, and some of them may fold into stable G-quadruplex (G4) structures in vitro. The stabilization of mtDNA G4s with potent small-molecule ligands in cancer cells may potentially interrupt mitochondrial metabolism such as impairing the oxidative phosphorylation system (OXPHOS) in ATP synthesis to cause energy deficiency. Therefore, mtDNA G4s have been an emerging drug target for chemical biology and anticancer study. Nonetheless, the development of potent ligands specifically targeting mitochondria and interacting with mtDNA G4s in living cells remains a challenge. This largely limits the feasibility to understand the mechanism of actions targeting mitochondria and mtDNA G4s for drug discovery. Herein, we designed and synthesized several new mitochondria-targeting small molecules that bind to mtDNA G4s in melanoma cancer cells (A375) to cause mitochondrial metabolism alternation. Among the ligands, B1N was found to be the most potent one to downregulate the expression of some critical mitochondrial genes and proteins, inhibit ATP synthesis, and substantially induce metabolism reprogramming to upregulate glycolysis. Moreover, the combination therapy study of 1.75 μM B1N with a clinical BRAF inhibitor (Vemurafenib, 0.2 μM) showed synergistic effects (CI = 0.67) against A375 cells. This new combined treatment significantly downregulates ATP production and glycolysis and induces acute senescence. The present study demonstrates an innovative and effective combination therapy strategy utilizing mitochondrion-targeting ligands and clinical inhibitors against melanoma.

The epidermal growth factor receptor (EGFR) mediates signal transduction by triggering downstream phosphorylation to regulate cell proliferation. However, the complexity of the cellular environment has limited in situ structural investigations of membrane proteins within their native context. Here, we present a proof-of-concept study integrating protein cage labeling with cryo-electron tomography (cryo-ET) to directly visualize receptor assemblies on the native membrane. Using EGFR as a model system, we demonstrate that the protein cage can associate with multiple EGFR molecules, thereby inducing their oligomerization. The distance between neighboring EGFRs within these assemblies was measured to be 7.1 ± 1.2 nm. Furthermore, we validated the functional relevance of this system by showing that protein cage-induced EGFR assemblies were accompanied by enhanced ligand-independent phosphorylation. In summary, our results establish the feasibility of using protein cage-labeling for the induction and in situ structural analysis of membrane protein oligomerization.