ATP citrate lyase (ACLY) is a pivotal enzyme implicated in cancer cells’ aberrant survival and proliferation. However, its precise role in tumor endothelial cells (TECs) in tumor angiogenesis and malignant progression remains poorly defined. Our previous studies have identified a high distribution of ginsenoside Rd in TECs, yet the mechanism underlying its anti-angiogenic properties has not been elucidated. Here, we clarify the direct regulatory effects of ACLY in TECs and explain how Rd reprograms the ACLY metabolic pathway in TECs. Systemic Acly silencing in tumor models resulted in suppressed angiogenesis and tumor progression. Importantly, TEC-specific Acly knockdown directly disrupted pathological vascularization and exerted comparable inhibitory effects on tumor growth. Metabolomic profiling revealed that ginsenoside Rd reduced nuclear acetyl-coenzyme A (acetyl-CoA) levels by down-regulating ACLY expression, thereby inhibiting the transcription of angiogenesis-related factors through reduced acetylation of histone H3 (but not H4). Mechanistically, ginsenoside Rd promotes the ubiquitin-mediated degradation of ACLY by targeting and suppressing ubiquitin-specific peptidase 13 (USP13). This study not only establishes a mechanistic framework for the development of ACLY-centric therapeutic strategies to reprogram pathological vasculature but also identifies the natural compound Rd as a pharmacological modulator of TECs metabolism via targeting the USP13–ACLY axis.
Conventional antibody–drug conjugates (ADCs) serve as promising targeted delivery systems that harnesses monoclonal antibodies to selectively deliver potent cytotoxic agents to tumor cells. However, clinical translation remains hindered by key challenges, including tumor heterogeneity, drug resistance, on-target off-tumor toxicity, and manufacturing complexity. To address these limitations, the field has evolved beyond classical ADC designs toward a diverse array of novel conjugates (XDCs). On the targeting side, extensive exploration of various carrier moieties (X) and bispecific strategies aims to overcome tumor heterogeneity and reduce off-tumor toxicity. On the payload side, beyond cytotoxic agents with distinct mechanisms, alternative payload formats, such as degraders, immune stimulators, and oligonucleotides, are being increasingly applied to further lower dose-limiting toxicities. These advances have given rise to platforms including bispecific ADCs, dual-payload ADCs, immune-stimulating antibody conjugates, degrader-antibody conjugates, antibody–oligonucleotide conjugates, peptide–drug conjugates, aptamer–drug conjugates, small molecule–drug conjugates, cell–drug conjugates, and virus-like drug conjugates. Leveraging their unique features, these architectures hold promise for improved therapeutic efficacy. This review highlights recent progress in XDCs design strategies and offers perspectives on future directions for next-generation targeted cancer therapies.
Specific and effective treatment of solid tumors has long been a challenge in cancer therapy. Antibody–drug conjugates (ADCs) represent a crucial advancement in precision oncology, with expanding applications and growing attention year by year. Traditional ADCs rely on monoclonal antibodies generated through animal immunization, followed by screening, sequencing, and recombinant expression—a process that is experimentally complex, time-consuming, and costly. In this study, we employed deep learning-based computational design methods to de novo design target-binding proteins specific for the tumor-associated antigen NECTIN-4. The designed NECTIN-4 mini-protein binder exhibited great specificity and thermal stability. The binding affinity was optimized from 0.262 μmol/L to 8.74 nmol/L through partial diffusion. Based on this, we further engineered the mini-protein binder by conjugating it with microtubule inhibitor VcMMAE to produce mini-protein drug conjugates (MPDCs) capable of targeted killing of NECTIN-4-positive tumor cells. We conducted a detailed evaluation of the anti-cancer activity of these MPDCs using cancer cells, organoids, and mouse xenograft models. Treatment experiments in xenograft models demonstrated that MPDCs exhibited good biological safety, with tumor growth being completely inhibited at an injection dose of 5 mg/kg. These designed MPDCs offer a new approach for preparing protein–drug conjugates for the precision treatment of solid tumors.
Obesity-related adipose tissue (AT) dysfunction is closely associated with the development and progression of metabolic dysfunction-associated steatotic liver disease (MASLD), the most prevalent chronic liver disease. Emerging evidence highlights that the AT-liver crosstalk is mediated by AT-derived extracellular vesicles (EVs) that exacerbate the progression of MASLD during obesity. During obesity development, AT undergoes pathological remodeling characterized by hypertrophic adipocytes, pro-inflammatory macrophages, dysfunctional adipose-derived stem cells (ADSCs), and increased cellular heterogeneity, which collectively alter the composition and function of secreted EVs. AT-derived EVs enter the circulation and transport bioactive cargo, including microRNAs (miRNAs), mtDNA, proteins and lipids to the liver, where they can modulate hepatocellular metabolism, liver inflammation and fibrogenesis. This review outlines the current state of knowledge on AT-derived EVs during MASLD, emphasizing the dynamic changes in AT under metabolic stress as well as the interactions between AT-derived EVs and the liver. We further explore the potential of EVs and their associated molecules as noninvasive biomarkers and potential therapeutic targets and underscore that understanding AT-liver communication via EVs offers perspectives for precision medicine in MASLD.
Securinine (1) and its three stereoisomers, allosecurinine (2), virosecurinine (3) and viroallosecurinine (4), can be produced in the medicinal plant Flueggea suffruticosa. Their stereochemistry critically impacts biological activity: only securinine acts as a potent GABAA receptor antagonist and was once clinically used as a central nervous system stimulant. However, the mechanism underlying their stereochemical control has remained elusive. Here, through isotope labeling, we elucidated that (±)-menisdaurilide and (±)-aquilegiolide serve as key intermediates. They can non-enzymatically couple with Δ1-piperideine to form neosecurinane alkaloids, subsequently undergoing intramolecular rearrangement to generate 1–4. Notably, the C6–OH configuration of (±)-menisdaurilide and (±)-aquilegiolide serves as a gatekeeper for controlling selective production of levorotatory products 1/2 and dextrorotatory products 3/4. To identify enzymes governing this stereoselective formation of C6–OH, we employed an isotope-guided spatial transcriptomic strategy, which revealed a critical spatial discrepancy: biosynthesis of securinine and its stereoisomers occurs in stems, whereas they accumulate in roots. Leveraging this spatial localization insight, we ultimately identified three stem-preferentially expressed ketoreductases that mediate stereoselective biosynthesis of these alkaloids. This study comprehensively delineates the mechanistic basis for stereochemical control of securinine and highlights the utility of the isotope-guided spatial transcriptomic strategy in efficiently identifying biosynthetic genes of plant natural products.
Spinal cord injury (SCI) triggers irreversible neurological dysfunction driven by sequential primary and secondary insults, with neuronal death and neuroinflammation as the central pathological determinants. Despite advances in neuroregeneration research, an incomplete understanding of the molecular regulators underlying these processes limits the development of effective SCI treatment strategies. Post-translational modifications (PTMs) function as critical “dynamic molecular switches” that spatiotemporally regulate protein function and signaling cascades, shaping the SCI microenvironment. Emerging evidence suggests that interdependent PTM networks—rather than isolated modifications—regulate the balance between deterioration and repair. This review synthesizes current insights into seven major PTMs implicated in neuronal death and neuroinflammation after SCI, emphasizing their cooperative crosstalk and the methodological challenges in decoding their temporal dynamics. We propose a “spatiotemporally regulated PTM network” framework to guide therapeutic interventions, highlighting multiplex PTM modulation as a promising strategy to enhance neuronal survival, reduce neuroinflammation, and promote functional recovery after SCI. This review deepens the understanding of PTM crosstalk in SCI pathology and provides new perspectives for developing novel PTM detection and clinical therapeutic strategies.
The clinical translation of ex vivo CAR-T therapy is constrained by its complex manufacturing processes, limited treatment accessibility, and the toxicities associated with lymphodepleting chemotherapy. In vivo CAR-T strategies, which utilize viral or non-viral vectors to reprogram a patient’s T cells directly in situ genetically, offer a promising alternative. This review provides a systematic examination of the critical barriers impeding the advancement of in vivo CAR-T therapy. We first summarize the factors influencing the targeting ability of both the delivery system and the CAR molecule. As for the vectors, we detail how the composition and physicochemical properties of LNPs shift their tropism from the liver to the spleen and provide a critical comparison of active targeting strategies based on different T-cell surface antigens. As for the CAR molecule, target selection is primarily dictated by the intended disease. Furthermore, we discuss the determinants of poor CAR-T cell persistence, including unstable mRNA payloads and exhaustion-associated signaling pathways. Addressing the challenges of solid tumor treatment, we dissect the multifaceted role of the tumor microenvironment. Finally, this review illustrates the mechanisms underlying overactivation and immunogenicity triggered by both viral and non-viral vector systems.
Conditionally active antibody‒drug conjugates (ADCs) can be achieved through antibody engineering and/or linker-payload design to achieve context-dependent activation, addressing the limitations of conventional ADCs such as premature payload release, inefficient tumor penetration, and on-target, off-tumor toxicities. Antibody surfaces can be modified with steric shields, site-specific conjugation, or reversible masking groups to further limit off-target binding. Linker-payloads are masked in circulation through prodrug strategies, such as multi-step or logic-gated linkers, releasable caps, or bioorthogonal chemistries. These strategies confine cytotoxic drugs to the tumor microenvironment (TME), enhancing intratumoral drug concentration, expanding the therapeutic window, and reducing systemic toxicity. Early clinical studies of conditionally active ADCs targeting ROR2, AXL, and CD71 have demonstrated promising safety and efficacy. This review discusses the underlying mechanisms, antibody engineering and linker-payload design strategies, and clinical progress of conditionally active ADCs, and considers future avenues to improve tumor selectivity and therapeutic performance.
Imbalance of copper homeostasis leads to cancer progression, while its molecular basis and treatment approaches in gastric cancer remain elusive. Herein, MYC proto-oncogene and kinesin family member 18B (KIF18B) were identified as transcriptional suppressors of metallothionein (MT) genes, thereby driving copper-mediated aggressiveness and ferroptosis resistance of gastric cancer. Mechanistically, KIF18B was stabilized via p62-mediated autophagic degradation of E3 ubiquitin ligase cullin 3 (CUL3), which activated MYC via physical interaction to repress expression of copper storage genes MT2A and MT1X, resulting in elevation of free copper, subsequent up-regulation of pyruvate carboxylase (PC) and ferritin heavy chain 1 (FTH1), increase of tricarboxylic acid cycle for energy production, and decrease of ferroptosis in gastric cancer. KIF18B facilitated growth or aggressiveness and inhibited ferroptosis of gastric cancer cells via increasing MYC activity. An inhibitory peptide was established to block interplay between KIF18B and MYC, which suppressed growth, invasion, or metastasis and induced ferroptosis of gastric cancer. High KIF18B, MYC, PC, or FTH1 expression was associated with poor prognosis, while low CUL3, MT2A, or MT1X levels were linked to unfavorable outcome of gastric cancer patients. These findings suggest that activation of KIF18B‒MYC axis by autophagic CUL3 degradation promotes copper imbalance essential for gastric cancer progression.
Estrogens impair bile acid (BA) homeostasis, supporting the development of intrahepatic cholestasis of pregnancy (ICP). ICP is characterized by elevated serum BA levels, pruritus, and an increased risk of adverse perinatal outcomes. The constitutive androstane receptor (CAR) regulates genes involved in xenobiotic and BA detoxification. Previous studies suggest that CAR activation is protective in bile duct ligation–induced cholestasis and that the main genes involved in BA elimination are downregulated in ethinylestradiol (EE)-induced cholestasis in mice. Here, we examined whether CAR ligands could ameliorate cholestasis and prevent liver injury in an EE-induced cholestasis model using biochemical, transcriptomic, and BA metabolomic analyses. We demonstrate that estradiol significantly downregulates and inhibits CAR in the liver. Consistently, EE significantly suppresses the expression of hepatic CAR target genes. The murine CAR agonist TCPOBOP partially restores the expression of selected detoxification genes, improves plasma alkaline phosphatase activity, and decreases hepatic BA accumulation. In humanized PXR–CAR–CYP3A4/3A7 mice with EE-induced cholestasis, MI763F, a novel and potent human CAR agonist, recapitulates the effects of TCPOBOP on key genes involved in BA metabolism and the BA metabolome, and significantly reduces hepatic BA accumulation. These results demonstrate that CAR ligands counteract EE-mediated disruption of genes involved in BA homeostasis.
Liver fibrosis is a pressing clinical challenge, poses a huge threat to human health. However, there is no approved therapeutic medication to treat this devastating disease. Using a drug repositioning or drug rescue (DDDR) strategy, we identified the anti-analgesic agent flupirtine as a potential anti-liver fibrosis candidate through cell-based high-throughput screening of the FDA-approved drug library. Treatment with flupirtine reversed TGF-β1-induced fibrogenesis and ameliorated liver fibrosis in CCl4 or DDC diet-treated fibrotic mice. Asparagine synthase (ASNS) was identified as the direct target of flupirtine. Flupirtine interacted with the residues Asp401 and Arg404 of ASNS, thereby inhibiting its enzymatic activity. Importantly, ASNS levels were increased in activated hepatic stellate cells (acHSCs) and in the livers of fibrotic mice and humans, and are positively correlated with the severity of liver fibrosis. Mechanistically, inhibition or deletion of ASNS to increase L-aspartate levels reversed TGF-β1-induced Ca2+ overload in mitochondria, leading to suppression of calpain 1/complex I/mitochondrial reactive oxygen species (mtROS) axis, thereby improving mitochondrial quality and enhancing mitochondrial oxidation, ultimately counteracting liver fibrosis. Our study identified ASNS as a potential therapeutic target for treating liver fibrosis, flupirtine counteracted liver fibrosis in mice by suppressing ASNS in acHSCs, thereby blocking mitochondrial Ca2+/calpain 1/complex I/mtROS axis.
Pathological cardiac hypertrophy and heart failure remain mechanistically incompletely defined despite their clinical significance in cardiovascular disease. While ADP-ribosyltransferase cholera toxin-like (ARTC) enzymes modulate membrane protein function and downstream signaling via post-translational modifications, their role in cardiac pathology remains unexplored. Herein, we investigated the regulatory involvement of ADP-ribosyltransferase 3 (ART3) in myocardial remodeling. Cardiomyocyte-specific ART3 knockdown and overexpression murine models were established using adeno-associated virus serotype 9 (AAV9), with RNA-sequencing employed to profile ART3-dependent transcriptional responses in pathological cardiac hypertrophy. Functional validation was performed in complementary in vitro and in vivo hypertrophy models, complemented by immunoprecipitation-coupled mass spectrometry to identify ART3 substrates. ART3 was found to be cardiomyocyte-enriched and transcriptionally downregulated during progressive myocardial remodeling and heart failure. Cardiomyocyte-specific ART3 knockdown 3 accelerated the decompensatory transition of cardiac hypertrophy, whereas ART3 overexpression exerted significant anti-hypertrophic and anti-remodeling effects. Mechanistically, ART3 preserved cardiomyocyte mono-ADP-ribosyltransferase activity, catalyzing the site-specific mono-ADP-ribosylation (mADPr) of Integrin Subunit Alpha 7 (ITGA7) at residue R129. This post-translational modification was requisite for integrin signaling pathway activation, which mediated the observed cardioprotective effects. Collectively, these findings establish ART3 as a novel regulator of pathological cardiac hypertrophy by modulating ITGA7 via mADPr, highlighting its therapeutic potential as a target for heart failure intervention.
The processing of traditional Chinese medicine (TCM) is a critical process that upholds tradition and determines medicinal efficacy. The mechanisms underlying changes in TCM efficacy resulting from processing are currently a key focus of TCM research. Advances in supramolecular chemistry have significantly advanced the understanding of the mechanisms governing the efficacy of TCM complex chemical systems. Both the active ingredient basis and the adjuvant materials used in processing can be regarded as supramolecular units. Processing guides the TCM supermolecules to change structure through covalent forces and self-assemble non-covalently, forming soft matter aggregates that enable selective recognition, transformation, and assembly of components. These aggregates autonomously steer the entire system toward enhanced efficacy and reduced toxicity, thereby revealing the TCM's true active substance foundation. This paper highlights that the dynamic chemical system formed via supramolecular self-assembly during TCM processing can be likened to an “imprinting template” with memory function, which underlies the TCM “holistic” effects—characterized by multiple components, pathways, and targets. This study aims to provide valuable insights into TCM processing based on supramolecular development, bridging the gap between supramolecular science and TCM. Consequently, it offers profound inspiration for designing next-generation smart drug delivery systems, responsive biomaterials, and adaptive chemical systems.
Hepatic inflammation and gut barrier dysfunction are core pathologies of alcohol-associated liver disease (ALD), yet effective therapies remain limited. Pueraria lobata polysaccharide (PLP1), a key active constituent of P. lobata Radix, has demonstrated hepatoprotective potential; however, its underlying mechanisms remain poorly understood. In this study, we sought to elucidate the therapeutic mechanism of PLP1 against experimental ALD by integrating proteomics, metabolomics, and gut microbiota analyses. We demonstrated that PLP1 initially functions through a microbiota-dependent pathway, transferable by fecal microbiota transplantation (FMT), to restore gut barrier integrity and suppress the gut‒LPS‒TLR4 inflammatory axis. Furthermore, PLP1 exerted a direct, microbiota-independent effect, maintaining its therapeutic efficacy in antibiotic-depleted mice. Mechanistically, PLP1 directly and specifically targets CXCL1, promoting its ubiquitin-mediated proteasomal degradation as confirmed by biophysical assays. The functional necessity of this interaction was definitively established via comprehensive genetic manipulation of CXCL1 in vitro and in vivo. Specifically, CXCL1 overexpression reversed PLP1’s benefits, whereas CXCL1 knockdown mimicked its effects and occluded any additional benefit from PLP1. Overall, PLP1 ameliorates ALD by independently targeting both extrinsic and intrinsic inflammatory triggers. This study reveals the multifaceted pharmacology of a natural polysaccharide and validates a dual-pronged therapeutic strategy for ALD.
Targeted activation of the stimulator of interferon genes (STING) signaling pathway represents a promising strategy to counteract immunosuppressive tumor microenvironments (TME) and elicit anti-tumor immunity. However, clinical translation of STING agonists has been strongly hindered by the challenges in achieving tumor-targeted STING activation. Here, we report a facile and efficient supramolecular self-assembly strategy for tumor-targeted STING activation of α-mangostin (MGT), a natural non-cyclic dinucleotide STING agonist. MGT can self-assemble into uniform supramolecular nanoparticles (MGT-SNPs) with 100% drug loading, driven by its inherent amphiphilic xanthone structure via multiple non-covalent interactions. The resulting MGT-SNPs (∼186.50 nm, negatively charged) demonstrate excellent colloidal stability, pH-responsive drug release, and enhanced cellular uptake in vitro. Furthermore, MGT-SNPs exhibit sufficient circulatory stability, enabling efficient tumor accumulation and cytosolic delivery via the enhanced permeability and retention (EPR) effect. Leveraging these favorable properties, MGT-SNPs enhance STING activation both in vitro and in vivo, effectively remodeling TME. Consequently, MGT-SNPs trigger robust anti-tumor immunity in melanoma-bearing mice following either intratumoral or intravenous administration. Collectively, this work showcases a supramolecular self-assembly strategy based on the intrinsic structural properties of a STING agonist for tumor-targeted STING activation, which eliminates carrier-related toxicity and offers a clinically promising approach for STING-targeted cancer immunotherapy.
Cancer remains a major global health challenge, driving intensive research into novel therapeutic strategies. Oncolytic viruses (OVs) including herpesviruses, adenoviruses and echoviruses, have emerged as promising agents for cancer treatment, with several achieving regulatory approval. These viruses selectively lyse tumor cells and stimulate antitumor immunity. Nevertheless, their efficacy is constrained by limitations such as neutralization by serum factors and tumor cell resistance. Concurrently, epigenetic inhibitors modulate gene expression to suppress tumor proliferation and reshape immune responses. Recent advances highlight the strong synergistic potential of combining epigenetic inhibitors with oncolytic viruses. This strategy enhances viral replication and tumor cell killing, reprograms the tumor microenvironment, and ultimately improves therapeutic efficacy. Consequently, the integration of epigenetic modulators with OV therapy represents a promising frontier in oncology. This review comprehensively examines epigenetic inhibitors, with emphasis on their combined application with oncolytic viruses for cancer treatment.
With the cross-integration of nanotechnology and immunology, nanomaterials have shown great potential in tumor treatment. This review begins with the nanomaterials have unique advantages in reshaping the tumor immune microenvironment (TIME), including improving targeting, enhancing stability, increasing delivery efficiency, achieving controllable release, and endowing a multi-functional integrated platform. We then systematically reviews the recently research progress on the multi-mechanism regulation of antigen-presenting cells (APCs) by nanomaterials: (1) In the aspect of dendritic cell activation, we focus on the breakthrough achievements such as the activation of immune responses through the generation of immune signal complexes and the promotion of tumor neoantigen presentation; (2) In the field of macrophage reprogramming, we analyze the applications of nanomaterials in inducing macrophage polarization, improving the anti-tumor phagocytosis of macrophages, blocking macrophage autophagy, and enhancing the synergy of immune cells; (3) In the regulation of B cells, we explore the mechanism by which nanomaterials enhance the germinal center response and thereby exert anti-tumor immune effects. This article further discusses key challenges in clinical translation, including the dynamic fate of nanomaterials in complex TIME and the balance between immunogenicity and efficacy, and proposes future directions for accelerating clinical translation through computer-aided design (such as AI prediction of nanomaterial-immune cell interaction networks) and organ-on-a-chip evaluation systems. This review provides a theoretical framework and technical route for the development of next-generation tumor immunonanomedicines.
Mesangial proliferative glomerulonephritis (MsPGN) is a common cause of end-stage renal disease, characterized by mesangial cell proliferation within glomeruli. Mesangial cell activation triggered by inflammation is a key factor in the development of MsPGN. However, effective therapeutic strategies targeting this process are still limited. Here, we uncovered, for the first time, the direct effects of chlorogenic acid (CGA), a naturally occurring small-molecule compound with anti-inflammatory and antiproliferative properties, on mesangial cells in an anti-Thy1 nephritis animal model. A multi-dimensional pharmacological platform integrating laser microdissection-coupled glomerular proteomics affinity deconvolution, surface plasmon resonance, molecular dynamics, and enzyme assays identified Ras-related C3 botulinum toxin substrate 1 (RAC1) as the direct target of CGA. Mechanistically, CGA competitively binds to the LYS15, PRO33, and THR34 amino acid residues—located within residues 57–65 of the GTP/GDP-binding domain of RAC1, inhibiting its activation and subsequently reducing AKT phosphorylation while suppressing Thrombospondin-1 secretion from mesangial cells—a key ligand for macrophage CD36 receptors. This interaction deactivates macrophages and lowers the levels of inflammatory cytokines, including TNFα, IL1β, and IL6. Significantly, as a novel natural RAC1 inhibitor, CGA disrupts mesangial-macrophage crosstalk by dual suppression of regional immunity and cellular proliferation, thus conferring renal protection in MsPGN models. Our findings highlight CGA as a promising pharmacotherapy, offering a mechanism-driven, natural product-based strategy to mitigate MsPGN progression.
Enterovirus D68 (EV-D68) is an emerging respiratory pathogen, with severe cases developing into acute flaccid myelitis, a paralytic condition. Despite growing concern, no approved antivirals currently exist for EV-D68, underscoring the urgent need for therapeutic discovery. Here, we report the development of a high-throughput, phenotypic-based assay to screen 7986 compounds across nine diverse compound libraries for EV-D68 antivirals, on two EV-D68-susceptible cell lines (RD and H1299). The screen identified GW406108X as a potent post-entry inhibitor of EV-D68 infection (EC50: 1.804 μmol/L). Mechanistic studies using luciferase replicon assays, siRNA knockdowns, and drug-resistant mutant generation suggest that GW406108X targets the autophagy pathway through ULK1/2 inhibition. Transmission electron microscopy and fluorescence bioimaging demonstrate a significant reduction in autophagosome formation in treated, infected cells. This disruption likely impairs the virus’s ability to exploit autophagy, which may in turn hinder replication organelle formation and non-lytic virion release, leading to reduced viral replication. Preclinical evaluations showed that GW406108X demonstrates strong antiviral efficacy without detectable cytotoxicity. These findings reveal a previously uncharacterized antiviral mechanism and position GW406108X as a promising candidate for antiviral therapeutic development. Findings from this study expand the current antiviral landscape for enteroviruses and represent a significant step toward clinical intervention for EV-D68 and potentially related viral pathogens.
