Silver ions (Ag+) have long been employed as natural antimicrobial agents, yet their precise mechanism of action remains unclear. In this study, we show that Ag+ displays strong antibacterial activity against Staphylococcus aureus (S. aureus), including methicillin-resistant strains (MRSA). Using chemoproteomic analysis, we identified MurB, MurC, and MurD as direct coordinate covalent targets of Ag+ in S. aureus, with binding occurring at cysteine residues Cys224, Cys368, and Cys221, respectively. This interaction leads to a reduction in MurB and MurD expression and inhibits MurC enzymatic activity, ultimately disrupting peptidoglycan synthesis and compromising bacterial cell wall integrity. Consequently, Ag+ treatment results in bacterial membrane leakage, altered membrane potential, and inhibited biofilm formation. Additionally, Ag+ reduces bacterial adhesion and invasion, alleviating the inflammatory response in host cells. Notably, Ag+ exhibits a low resistance frequency compared to conventional antibiotics, underscoring its potential as an effective antimicrobial agent. Its distinct mechanism of action and reduced likelihood of resistance development indicate that it may serve not only as an effective therapeutic strategy, but also as a probe for elucidating the mechanism of bacterial peptidoglycan biosynthesis. These results offer new insights for the development of antibiotics targeting Mur family proteins.

Triple-negative breast cancer (TNBC) represents the most aggressive breast cancer subtype with limited therapeutic options and poor prognosis, highlighting an urgent need to identify novel metabolic vulnerabilities and prognostic biomarkers to improve patient outcomes. While glutamine metabolism has been implicated in cancer, the specific roles of glutamine metabolism-related genes (GMRGs) in TNBC remain poorly understood. Herein, through an integrating analysis encompassing multi-omics bioinformatics, consensus clustering and tumor microenvironment (TME) analysis, we established a glutamine metabolism-based prognostic classification for TNBC patients, which correlates with distinct survival outcomes and TME features. Furthermore, we identified ALDH18A1, one of the GMRGs that encodes P5CS for proline synthesis, as a novel prognostic biomarker and oncogenic driver. ALDH18A1 is overexpressed in TNBC and associated with larger tumor size, lymph node metastasis and poor survival, as well as an immunosuppressive TME. In vitro experiments confirmed ALDH18A1 activated the AKT/mTOR signaling pathway, promoted the proliferation, migration and invasion of TNBC cells, and increased proline synthesis. Computational drug screening predicted six compounds with potential efficacy against ALDH18A1-high tumors. Collectively, our findings demonstrate that reprogramming of glutamine metabolism plays a crucial role in the malignant progression of TNBC and provide translational insights for precision metabolomic-immunotherapeutic strategies in ALDH18A1-high TNBC.

Background: Non-small cell lung cancer (NSCLC) remains a significant health challenge, and the KRAS mutation plays a critical role in its development, especially KRASG12C. Rosmarinic acid (RA), a natural polyphenolic compound, has demonstrated robust anti-cancer activities. However, the precise molecular mechanisms underlying its anti-tumor effects in NSCLC remain poorly understood. Methods: We assessed RA's anti-tumor effects in vitro/in vivo NSCLC models. Activity-based protein profiling (ABPP) coupled with bioorthogonal click chemistry identified RA's direct molecular targets. Western blot and other techniques analyzed KRAS/AKT/ERK signaling, plus cell cycle/apoptosis molecule expression. The effect of RA on tumor suppression and the ability of tumor cells to evade macrophages in an NSCLC mouse model with KRASG12C mutation was evaluated. Results: RA significantly inhibited NSCLC cell proliferation and induced apoptosis by modulating the KRAS/AKT/ ERK signaling cascade. ABPP analysis revealed that RA directly binds to KRASG12C at the mutant cysteine-12 residue. Functional studies confirmed that RA-mediated cell cycle arrest and apoptosis depend on KRASG12C modulation. In KRASG12C-mutant mouse models, RA markedly suppressed tumor growth and reduced macrophage evasion by tumor cells. Conclusions: RA acts as a novel KRASG12C inhibitor that directly targets the mutant cysteine-12 residue, suppressing NSCLC progression through inhibition of the KRAS/AKT/ERK pathway and enhancement of anti-tumor immune activity. These findings highlight RA's therapeutic potential for KRASG12C-driven NSCLC and offer new insights for the development of targeted cancer therapies.

Triple-negative breast cancer (TNBC) is an aggressive subtype lacking targeted therapies due to the absence of hormone receptors and HER2 expression, resulting in poor clinical outcomes and limited treatment options. Identifying novel vulnerabilities is therefore critical to advancing TNBC therapeutics. Mitochondrial metabolism has emerged as a key regulator of cancer cell survival and proliferation, with serine hydroxymethyltransferase 2 (SHMT2) playing a central role in mitochondrial one-carbon metabolism by supplying one-carbon units for nucleotide biosynthesis and maintaining redox homeostasis. Despite its established importance in cancer metabolism, the functional role and therapeutic potential of SHMT2 in TNBC remain underexplored. Here, we demonstrate that gambogic acid (GA), a natural product with reported anticancer properties, exerts potent and selective cytotoxicity against TNBC cells by covalently targeting SHMT2. GA binds specifically to the critical cysteine residue Cys241, inhibiting SHMT2 enzymatic activity and disrupting mitochondrial function. This leads to bioenergetic collapse, activation of the Nrf2/HO-1 axis, iron overload, and induction of ferroptosis, a non-apoptotic form of cell death increasingly recognized for its therapeutic potential. Our integrative chemoproteomic and mechanistic studies reveal a novel SHMT2-mitochondria-Nrf2/HO-1-ferroptosis axis driving GA's anti-TNBC activity. Moreover, SHMT2 overexpression in TNBC correlates with tumor aggressiveness and poor prognosis, underscoring its role as a metabolic oncogene and promising drug target. These findings establish GA as a novel covalent SHMT2 inhibitor and provide a new framework for exploiting metabolic vulnerabilities to overcome TNBC treatment resistance.

Introduction Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease characterized by chronic inflammation and fibroblast activation, with limited treatment options. The bioactive diterpenoid andrographolide (AP) exhibits anti-inflammatory and antifibrotic properties, but its direct molecular targets and precise mechanism remain unclear. Objectives This study aimed to identify the primary functional target of AP and elucidate the molecular mechanism underlying its therapeutic effects against pulmonary fibrosis. Methods We employed activity-based protein profiling (ABPP) to identify covalent targets of AP in macrophages. Functional validation was performed using siRNA knockdown, enzymatic assays, molecular docking, and biophysical analyses. The therapeutic efficacy and target dependency of AP were evaluated in bleomycin-induced pulmonary fibrosis and LPS-induced acute lung injury mouse models, utilizing lung-specific Ptges3 knockdown. Results AP covalently bound to Cys58 of prostaglandin E synthase 3 (Ptges3), an allosteric site distinct from its catalytic and Hsp90-binding regions. This binding inhibited Ptges3 enzymatic activity, reduced prostaglandin E2 (PGE2) production, and disrupted the Ptges3-Hsp90 chaperone complex, leading to suppressed NF-κB signaling. Genetic knockdown of Ptges3 significantly attenuated the anti-inflammatory and antifibrotic effects of AP both in vitro and in vivo. Conclusions Our findings establish Ptges3 as a critical functional target of AP. AP attenuates pulmonary fibrosis through a dual mechanism involving covalent inhibition of Ptges3 and disruption of the Ptges3-Hsp90-NF-κB axis, highlighting AP as a promising therapeutic agent and Ptges3 as a novel druggable target for IPF.

Balancing short-term antibacterial needs with long-term anti-inflammatory effects remains a major challenge in wound healing. Multifunctional bioactive materials capable of both efficient antibacterial action and inflammation modulation represent a promising solution. However, the majority of traditional antibacterial biomaterials possess only a single antibacterial effect, and their synthesis and preparation are intricate, which might restrict their clinical transformation. Chlorogenic acid is a natural compound endowed with anti-inflammatory properties. However, it is beset by certain inherent drawbacks, including poor water solubility and limited bioavailability. To overcome these difficulties, we have fabricated multifunctional nanoparticles (chlorogenic acid-iron nanoparticles, CA-Fe NPs) through the co-assembly of chlorogenic acid and iron ions in a straightforward manner. We found that CA-Fe NPs exhibit excellent photothermal conversion performance in vitro. Upon near-infrared (NIR) irradiation, they exhibit potent broad-spectrum antimicrobial activity against Staphylococcus aureus, Escherichia coli, Candida albicans, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The CA-Fe NPs markedly reduced H2O2-induced reactive oxygen species levels and apoptosis in epithelial cells and suppressed lipopolysaccharide-induced M1 macrophage polarization in RAW 264.7 cells. Transmission electron microscopy results revealed enhanced bacterial membrane disruption by CA-Fe NPs under NIR irradiation, causing pronounced protein leakage. Transcriptomic analysis indicates that CA-Fe NPs combined with NIR disrupt the tricarboxylic acid cycle, cell-wall organization, and other metabolic processes. In vivo, within a methicillin-resistant Staphylococcus aureus-infected skin wound model, CA-Fe NPs maintained photothermal efficacy, effectively reduced serum levels of interleukin-1 beta, interleukin-6, and tumor necrosis factor alpha, and accelerated wound healing. These findings suggest that CA-Fe NPs are multifunctional materials with broad-spectrum bactericidal ability, antioxidant, anti-inflammatory, and wound-healing-promotion properties. These nanoparticles possess promising prospects for biomedical applications.

Ischemic stroke remains a major clinical challenge due to limited treatment options and the lack of effective neuroprotectants. Here, we identified a novel neuroprotective mechanism of rosmarinic acid (RosA), a natural phenolic compound, through precision targeting of the autophagy regulator BAG3. Using activity-based protein profiling, we demonstrated that RosA covalently bound to the Cys378 residue of BAG3, disrupting its interaction with the selective autophagy receptor P62. This disruption activated the P62/Keap1/Nrf2 signaling axis, attenuating excessive autophagic flux and reducing neuronal injury. Both in vitro oxygen-glucose deprivation/reoxygenation (OGD/R) and in vivo middle cerebral artery occlusion/reperfusion (MCAO/R) models confirmed that RosA significantly reduced autophagosome accumulation, infarct volume, and neurological deficits in a BAG3-dependent manner. BAG3 knockdown mimicked RosA’s effects and abolished RosA-induced autophagy regulation, highlighting BAG3 as the functional target. These findings not only elucidated the molecular mechanism of RosA but also proposed BAG3 as a promising therapeutic target for ischemic stroke intervention.

Aging is a well-recognized risk factor in cardiovascular diseases (CVDs), primarily due to its association with the gradual decline in cardiac function. This decline significantly influences the pathogenesis of common CVDs such as myocardial infarction and heart failure. Despite the existence of several proteomic atlases of the heart, the spatially resolved proteomic dynamics essential for understanding region-specific aging mechanisms in cardiac tissue remain incompletely characterized. In this study, we conducted a region-resolved quantitative proteomic profiling for various murine cardiac regions at three distinct stages of aging (3, 12, and 20-month-old), quantifying 6 650 proteins in the heart. Leveraging integrated bioinformatics and machine learning frameworks, we uncovered that FTL1 and SERPINA3K exhibit strong age-associated expression changes across all cardiac regions. Mechanistically, the knockdown of Ftl1 led to cardiomyocyte ferroptosis and senescence, phenotypes that were ameliorated by the ferroptosis inhibitor Ferrostatin-1. Furthermore, the depletion of Serpina3k exacerbated senescence and collagen deposition through the activation of the cGAS-STING-PERK axis, effects that can be reversed via the overexpression of Serpina3k or the knockdown of Sting. The protective effect of SERPINA3K was also demonstrated in vivo through AAV9-mediated cardiomyocyte-specific overexpression in middle-aged mice, which attenuated the cGAS-STING-PERK axis and mitigated age-related fibrosis. These results strongly demonstrated that FTL1 and SERPINA3K function as key regulators of cardiac aging. Collectively, this study provides a valuable region-resolved proteomic atlas of cardiac aging and identifies key protein regulators, thereby uncovering potential targets for cardio-protective interventions against age-related cardiovascular disorders.

Quercetin (QC) is a natural flavonoid with poor bioavailability and unclear precise mechanism in myocardial ischemia/reperfusion (MI/R) injury. We identified high mobility group box-1 protein (HMGB1) as a direct cysteine‑binding target of QC using IAA-yne-based chemical proteomics, which was further validated by cellular thermal shift assay, pull-down experiments, siRNA interference, and bio-layer interferometry experiments. Mechanistic studies revealed that QC binding to HMGB1 inhibits the activation of NF-κB signaling pathway, thereby reduced the release of pro-inflammatory cytokines. To overcome the limitation of QC's delivery, we innovatively engineered myocardial-targeted liposomal nanoparticles (PEG-PEP/QC NPs). Pharmacokinetic analysis revealed this NPs significantly increased the QC's plasma concentration, prolonged the half-life, and, crucially, enhanced the accumulation of QC in cardiac tissue. In the rat MI/R model, this targeted formulation alleviated cardiac dysfunction, suppressed inflammatory response, and reduced myocardial injury. Together, our findings highlight the promise of targeted nanodelivery systems for MI/R therapy.

Bacterial outer membrane vesicles (OMVs) garner significant attention in tumor therapy due to their unique ability to selectively target and eliminate tumor cells. However, tumor cells commonly overexpress CD47, a “don’t eat me” signal that interacts with signal regulatory protein alpha (SIRPα) on macrophages, thus evading immune clearance. Enhancing OMV-induced immunogenicity and reprogramming macrophages toward a pro-phagocytic phenotype while blocking the CD47-SIRPα pathway is essential. In this study, OMVs are engineered to express anti-CD47 antibodies on their surface and are combined with Y18 nanoparticles (NPs) to construct a biomimetic hybrid nanoplatform (Y18-OMV NPs). This dual-functional strategy integrates photothermal design and immune checkpoint blockade to achieve efficient photoimmunotherapy. Photothermal therapy (PTT) induces immunogenic cell death via endoplasmic reticulum stress (eIF2α-ATF4-CHOP pathway) and pyroptosis (NLRP3/Caspase-1/GSDMD axis), releasing damage-associated molecular patterns (DAMPs) to activate immune responses. Simultaneously, Y18-OMV NPs function as immune adjuvants to repolarize M2-like tumor-associated macrophages (TAMs) into the M1 phenotype, enhance macrophage-mediated phagocytosis, promote dendritic cell maturation, and activate T cell infiltration. This strategy also downregulates heat shock proteins (HSPs), reduces thermotolerance, and enhances PTT efficacy. Overall, this approach provides robust photothermal-immunotherapy synergy and offers a promising direction for CD47-targeted clinical tumor immunotherapy.

While Chlamydomonas is a widely used model organism, nuclear transgene expression in this system is well documented to face notorious difficulties. Here, we show that 5' untranslated region (UTR) introns can play a positive role in reducing gene silencing. By screening promoter-5' UTR complexes containing introns (PUTRis) from highly expressed genes, we identify many candidates that outperform the two most widely used promoters, HR and PSAD. Truncation analyses reveal that intron removal increases gene silencing to a similar extent as promoter deletion. These 5' UTR introns promote open chromatin configurations that support gene expression. Leveraging two identified PUTRis, we achieve robust expression of previously recalcitrant Chlamydomonas proteins and visualize the subcellular localization of over 100 such proteins. Taken together, our findings demonstrate the role of 5' UTR introns in reducing gene silencing while providing the research community with molecular tools for algal genetic engineering.

Solid tumors, especially pancreatic ductal adenocarcinomas (PDACs), activate quiescent fibroblasts into cancer-associated fibroblasts (CAFs) that generate dense desmoplastic stroma. This barrier restricts drug penetration and immune infiltration, promoting tumor progression. Here, we engineer Midkine (MDK)-targeting nanobody-functionalized extracellular vesicles (D4-EV) as a precision photoimmunotherapy platform. These vesicles selectively accumulate in the tumor microenvironment through MDK overexpression. Loaded with chlorin e6 (Ce6), Ce6@D4-EV induces immunogenic cell death upon light irradiation, triggering dsDNA release and cGAS-STING activation in tumor-associated macrophages. Concurrently, it reprograms CAFs, reduces extracellular matrix deposition, improves vascular perfusion, and alleviates hypoxia. This stromal-immune remodeling substantially enhances the therapeutic efficacy of immune checkpoint blockade, adoptive T cell therapy, and chemotherapy, leading to prolonged survival in multiple MDK-positive preclinical tumor models. The platform provides a promising strategy to overcome stromal barriers in desmoplastic tumors.

BACKGROUND:Sepsis, a life-threatening condition, arises from an aberrant or uncontrolled immune response of the host to severe infection. Timely and precise control of this inflammatory response is of paramount importance in sepsis prevention and constitutes the core of sepsis prevention efforts. Parthenolide (PA), recognized as an NF-κB inhibitor, has previously demonstrated its capacity to mitigate the inflammatory response. This study aimed to investigate whether and how PA can inhibit inflammation, thereby alleviating sepsis, and to profile functional target proteins using chemoproteomics strategy. METHODS:The cecal ligation and puncture (CLP) procedure was performed to induce a mouse model of sepsis, and lipopolysaccharide (LPS) was employed to establish a model of cellular inflammation in mouse mononuclear macrophage cells (RAW264.7) in vitro. Enzyme-linked immunosorbent assay (ELISA) and hematoxylin and eosin (H&E) staining were utilized to evaluate the therapeutic effects of PA on septic mice. In the in vitro experiments, streamlined cysteine activity-based protein profiling (SLC-ABPP) and proteomics techniques were employed to identify potential protein targets and molecular pathways responsible for the anti-inflammatory properties of PA. RESULTS:We demonstrated that PA could enhance the survival rate, reduce inflammatory cytokines release, ameliorate histopathological changes, and balance macrophage polarization in a sepsis model. Similarly, PA could effectively alleviate the inflammatory response in LPS-stimulated RAW264.7 cells. Chemoproteomics profiling revealed that the Trim33 protein was a potential covalent target of PA via cysteine engagement, and the expression changes of global proteins indicated by proteomics analysis suggested that PA mitigated inflammation by inhibiting the NF-κB pathway. Finally, through a series of molecular biology experiments, we confirmed that PA suppresses the NF-κB pathway by functionally binding to Trim33 and subsequently reducing the ubiquitin dependent degradation of Smad4, thereby exhibiting anti-inflammatory activity. CONCLUSION:Our findings demonstrated that PA significantly mitigated inflammation in sepsis by targeting the Trim33 protein and consequently suppressing ubiquitination on Smad4 as well inhibiting the NF-κB pathway, thus providing new insights into the clinical use of PA in treatment of inflammation related diseases.

KEY POINTS:A natural sesquiterpene lactone compound Eupalinolide B protected against diabetic kidney disease. Eupalinolide B targeted cell division cycle 37 protein and disrupted the interaction between cell division cycle 37 with heat shock protein 90. The renoprotective effects of Eupalinolide B were dependent on cell division cycle 37-mediated inflammatory pathways in diabetic kidney disease. BACKGROUND:Diabetic kidney disease (DKD) is a major complication of diabetes, driven by chronic inflammation throughout its initiation and progression. Developing effective novel therapeutics is urgently needed. Eupalinolide B, a natural small compound derived from Eupatorium lindleyanum DC ., has multiple bioactive properties, notably antitumor and anti-inflammatory activities. However, the therapeutic potential of Eupalinolide B for DKD remains unclear. This study was to investigate the potential effects, direct targets, and pharmacologic mechanisms of Eupalinolide B against DKD, and discover novel therapeutic targets in the progression of DKD. METHODS:The therapeutic effects of Eupalinolide B were assessed in high glucose-induced rat mesangial (HBZY-1) cells and db/db diabetic mice. Targeting and binding site engagement of Eupalinolide B was validated through activity-based protein profiling technology, pull-down assay, surface plasmon resonance analysis, and high-resolution mass spectrometry analysis. The target protein was knocked down to investigate its role in DKD-related inflammation and determine whether Eupalinolide B's renoprotective effects depend on the protein. The impact of Eupalinolide B on protein-protein interactions was examined using immunoblotting, immunohistochemistry, immunofluorescence, and coimmunoprecipitation assays. RESULTS:Eupalinolide B was observed to ameliorate the glomerular filtration dysfunction and histopathologic damage in db/db mice and suppress NF-κB and mitogen-activated protein kinase proinflammatory pathways in both high glucose-induced HBZY-1 cells and db/db mice. In addition, we found that Eupalinolide B directly bound to cysteine 64 and 234 residues of cell division cycle 37 (CDC37), the essential cochaperone of heat shock protein 90 (HSP90). Mechanistically, by targeting CDC37, Eupalinolide B disrupted the interaction between CDC37 and HSP90, consequently blocking downstream proinflammatory signaling upon high glucose induction in HBZY-1 cells. CONCLUSIONS:Eupalinolide B was identified as a novel CDC37-targeting agent with renoprotective and anti-inflammatory effects against DKD that functions by inhibiting CDC37-HSP90 interaction.

Iron (Fe) and copper (Cu) are vital micronutrients that regulate many critical physiological processes in the human body, with their homeostasis in the central nervous system (CNS) being essential for proper neuronal function. Disruptions in their metabolism and regulatory pathways have been associated with the pathogenesis of various forms of neurodegenerative diseases (NDDs) such as Alzheimer's disease (AD) and Parkinson's disease (PD). Despite growing research on metal homeostasis, the intricate molecular mechanisms that link iron and copper metabolism to the initiation and progression of NDDs remain insufficiently elucidated. In this review, we provide a systematic overview of the metabolic processes of iron and copper in the body and CNS, highlighting their interactions with many metal-binding proteins, including transporters, storage proteins, and important intrinsically disordered proteins (e.g., amyloid β-protein, tau, and alpha-synuclein) involved in NDDs. We further dissect the downstream effects of metal ion dyshomeostasis on cellular redox balance, neuroinflammation, autophagy, organelle interaction network, and cell death. Additionally, we discuss current therapeutic strategies aimed at targeting iron and copper dyshomeostasis, as well as the emerging role of artificial intelligence in this field of research. By integrating metal metabolism, metal-protein interactions, the effect of metal dyshomeostasis on downstream biological processes, and potential intervention strategies, this review serves as a comprehensive reference for understanding the pathogenesis of NDDs and offers new perspectives for developing effective therapeutics. Overall, this review underscores the significance of reinstating metal balance for the treatment of neurodegeneration.