Aloperine derivatives can promote the degradation of PD-L1 through different pathways and elicit antitumor immunity, but their direct targets remain unclear. In this study, 30 aloperine derivatives were synthesized and screened for their efficacy in reducing PD-L1 levels, and compound 7a was found to effectively promote PD-L1 degradation through the lysosomal pathway. Then, based on the DeepDTAGen deep learning model, lysosome-associated membrane protein 2 (LAMP2) was predicted to be a potential functional target of 7a. Multiple biochemical assays demonstrated that 7a induced PD-L1 degradation through the LAMP2-mediated lysosomal pathway, with a KD value of 5.58 μM. Furthermore, molecular docking and molecular dynamics simulations proposed a plausible binding mode of 7a with LAMP2. Therefore, the integrated computational-experimental strategy efficiently identified LAMP2 as a direct-binding functional target of 7a for the degradation of PD-L1 via lysosomal pathway, providing a reference case for the rapid discovery of target.

ABSTRACT Antibody‐antibiotic conjugates (AACs) provide a transformative platform for eradicating intracellular methicillin‐resistant Staphylococcus aureus (MRSA). However, current AACs largely follow antibody‐drug conjugates (ADCs) design principles and rely on host‐lysosomal proteases for activation, rendering them ineffective against extracellular (planktonic) bacteria. Furthermore, we demonstrate that high bacterial burden induces host‐lysosomal dysfunction, fundamentally compromising the efficacy of host‐dependent AACs against intracellular reservoirs. To address these limitations, we developed a pathogen‐centric AAC (AZO‐AAC) featuring an azobenzene‐based linker that is triggered by bacterial‐secreted azoreductase. This strategy shifts the activation mechanism from host‐cell machinery to the pathogen itself, enabling antibiotic release independent of host‐cell integrity. Surprisingly, our designed AZO‐AAC achieves nanomolar‐scale eradication of both planktonic and intracellular MRSA, reaching levels below the limit of detection in scenarios where traditional AACs fail. In murine models of peritonitis and septicemia, a single dose (60 mg/kg) of AZO‐AAC resulted in a six‐log reduction in bacterial burden and the preservation of normal tissue architecture. By decoupling activation from host‐cell status, this pathogen‐responsive platform provides a robust strategy for the targeted elimination of complex, multi‐niche MRSA infections.

Background Immune checkpoint blockade (ICB) therapy has emerged as a pivotal cancer treatment by activating antitumor immunity. However, its clinical efficacy remains limited in many patients, highlighting the need for combination strategies to overcome resistance. Inducing immunogenic cell death (ICD) represents a promising approach to remodel the immunosuppressive tumor microenvironment and improve ICB efficacy. Methods A high-throughput screen of a natural compound library identified potent ICD inducers. Polyphyllin II (PPII), a bioactive component from Paris polyphylla , was selected for further investigation. Its effects on ICD markers, tumor growth, and immune activation were evaluated in vitro and in vivo. Limited proteolysis-mass spectrometry was employed to identify the direct target of PPII, followed by mechanistic studies using molecular and immunological assays. Results PPII was identified as a potent ICD inducer, stimulating the release of high mobility group box 1, ATP, and calreticulin from tumor cells. PPII suppressed tumor growth and enhanced antitumor immunity by promoting dendritic cell maturation and antigen cross-presentation, leading to CD8 + T-cell activation. Mechanistically, PPII directly bound to glucose-regulated protein 75 (GRP75), enhancing endoplasmic reticulum-mitochondrial tethering, provoking endoplasmic reticulum stress and mitochondrial calcium overload, and promoting cytochrome c release and caspase-3 activation. This cascade ultimately triggered gasdermin E (GSDME)-mediated pyroptosis. Furthermore, PPII synergized with anti-programmed cell death protein 1 therapy by reprogramming the tumor immune microenvironment and promoting systemic antitumor immunity. Conclusion Our findings identify GRP75 as a novel therapeutic target for cancer immunotherapy and highlight PPII-driven immune reprogramming as a translatable strategy to potentiate ICB efficacy through the induction of immunogenic pyroptosis.

The rising global cancer burden underscores the urgent need for more effective drug development and personalized therapies. Conventional screening models, such as 2D cell lines and patient-derived xenografts, fail to adequately recapitulate the architecture, heterogeneity, and microenvironment of human tumors, limiting their clinical translatability. In response, human-derived biomimetic platforms have emerged. Organoids and organ-on-a-chip preserve key tumor genetics and stimulate dynamic, physiologically relevant microenvironments, whereas microtumors are distinguished by high biological fidelity. Microtumors uniquely retain the native tumor ecosystem, capture a broader spectrum of intratumoral heterogeneity, and, critically, maintain a functional immune microenvironment. Together, these systems enable drug screening that more faithfully reflects the clinical context, with strong potential to raise drug development success rates and support individualized therapy. This review consolidates the cutting-edge advancements and critical challenges associated with these models in drug development, precision medicine, and clinical translation. Furthermore, it envisions how Artificial Intelligence (AI) can drive its intelligent evolution, aiming to provide a robust evidentiary basis and practical reference for research and clinical practice, thereby propelling the field of precision oncology into a new era.

BACKGROUND:Despite revolutionizing cancer treatment, immune checkpoint therapy (ICT) shows limited efficacy, primarily due to the non-immunogenic and immunosuppressive microenvironments. Identifying small-molecule inducers of immunogenic cell death (ICD) from natural sources represents a compelling strategy to overcome this therapeutic barrier. OBJECTIVE:This study aimed to identify natural ICD inducers and characterize their mechanisms in potentiating ICT efficacy. METHODS:High-throughput screening of a natural product library was performed to identify candidate ICD inducers. Pulsatilla saponin D (PSD), a triterpenoid saponin isolated from Pulsatilla koreana, was selected for comprehensive mechanistic and therapeutic evaluation. ICD induction was evaluated by measuring the release of damage-associated molecular patterns (DAMPs). The direct intracellular target of PSD was identified by limited proteolysis-mass spectrometry (LiP-SMap). Downstream mechanistic alterations in mitochondrial dynamics, mitochondrial reactive oxygen species (mtROS) generation, and hallmarks of ferroptosis were comprehensively characterized. RESULTS:PSD robustly elicited the release of DAMPs, signifying enhanced immunogenic stress. In both in vitro and in vivo models, PSD treatment significantly inhibited tumor progression by promoting the maturation of dendritic cells (DCs) and enhancing antigen cross-presentation, which subsequently invigorated CD8⁺ T-cell responses. Mechanistically, PSD binds directly to SHP2 (Src homology region 2 domain-containing phosphatase 2), impairing DRP1 oligomerization and inducing mitochondrial dysfunction. This cascade leads to an accumulation of mtROS and accelerated lipid peroxidation, ultimately executing ferroptotic cell death. Notably, the combination of PSD with anti-PD-1 blockade demonstrated a synergistic antitumor effect, characterized by an increased infiltration of cDC1 and cytotoxic T cells, alongside a reduction in immunosuppressive populations. CONCLUSION:Our work implicates SHP2-mediated ferroptosis as a potentially targetable mechanism for cancer immunotherapy and supports PSD as a proof-of-concept lead compound and preclinical pharmacological probe for investigating combination strategies with ICT.

Covalent modification represents an effective strategy in drug discovery. We strategically constructed a series of novel 12N-substituted enone-aloperines through incorporation of an α,β-unsaturated carbonyl, as a covalent warhead, into the aloperine endocyclic scaffold, to improve their efficacy in suppressing PD-L1. Although both the key analogues 8e and 8k bond to the target proteins in a reversible covalent manner as expected, their mechanisms of down-regulating PD-L1 are completely different. Compound 8e featuring a 12N-trifluoromethyl benzenesulfonyl targets UCHL3 to accelerate the PD-L1 degradation through the proteasomal pathway, while 8k with a naphthalenesulfonyl bonds to MNK1, suppresses the p-eIF4E, thereby inhibiting the subsequent translation of PD-L1. Cluster analysis indicates that the electron cloud density and polarity on the 12N-side chain play a crucial role in matching distinct targets. These results demonstrate that enone-aloperines can act as reversible covalent inhibitors of MNK1 or UCHL3 to exert beneficial anti-tumor immune effects. Especially, the subtle structural modifications of small molecules can redirect mechanisms of action, highlighting a strategic approach for uncovering novel drug targets and expanding therapeutic opportunities, and thus are worthy of further attention.

Alveolar macrophages (AMs) are indispensable to prevent pulmonary alveolar proteinosis and clear inhaled pathogens. Receptor for activated C kinase 1 (RACK1) is a versatile adaptor protein that regulates multiple signaling pathways. Whether RACK1 is implicated in AM alterations remains elusive. Alveolar type 2 cells-derived granulocyte-macrophage colony-stimulating factor and autocrine transforming growth factor-β1 drive the transcription of Pparg, the gene encoding AM signature transcription factor peroxisome proliferator-activated receptor-γ (PPARγ). The regulation of PPARγ stability during AM development and maintenance remains unexplored. Here, we report that myeloid RACK1 deficiency results in the scarcity of mature AMs and pulmonary alveolar proteinosis. A mixed bone marrow chimera approach reveals a cell-intrinsic role of RACK1 in AM differentiation. Bulk RNA-sequencing indicates a considerable loss of AM identity, impaired PPAR signaling, but a largely unchanged Pparg messenger RNA (mRNA) level in the absence of RACK1. Indeed, myeloid deletion of Rack1 halts AM differentiation in vivo and blocks the ability of PPARγ agonist to induce AM-like cells in vitro. Mechanistically, RACK1 directly binds to and stabilizes PPARγ by preventing its ubiquitination and degradation. Moreover, myeloid RACK1 deficiency renders mice susceptible to Streptococcus pneumoniae infection.

A promising therapeutic approach in oncology involves immune checkpoint blockade (ICB), which stimulates anti-tumor immune responses. Nevertheless, the effectiveness of this treatment in clinical settings remains limited, underscoring the need for complementary strategies. Recent studies highlight the potential of type I interferon (IFN-I) inducers to reprogram the tumor microenvironment and enhance ICB outcomes. Herein, through high-content screening of a natural compound library, we identified daurisoline (DS), a bioactive alkaloid extracted from the Chinese herbal medicine Rhizoma Menispermi, as a potent inducer of IFN-I signaling. Our findings indicated that DS up-regulates interferon responses and pro-inflammatory cytokine expression in a TANK-binding kinase 1 (TBK1)-dependent manner. In vivo, DS exhibited marked tumor growth inhibition by activating dendritic cells, macrophages, and CD8 + T cells, thereby enhancing anti-tumor immunity. Utilizing the LiP-SMap approach, we identified low-density lipoprotein receptor-related protein 1 (LRP1) as the direct target of DS. Mechanistically, the binding of DS to LRP1 substantially disrupted lysosomal function, which subsequently triggered 5′-azacytidine-induced protein 2-mediated TBK1 activation and IFN-I production. Furthermore, DS demonstrated synergistic effects with anti-programmed death 1 therapy and a stimulator of interferon genes agonist by remodeling the immunosuppressive microenvironment. Collectively, our findings establish LRP1 as a novel therapeutic target for cancer immunotherapy and highlight DS-driven immune reprogramming as a translatable strategy to potentiate ICB efficacy.

Immune‐stimulating antibody conjugate (ISACs) incorporating STING agonists as payloads leverage both the targeting capability of the Fab region and the Fc region‐mediated tumor antigen‐dependent immune activation. Herein, a novel class of ISACs is reported, generated by engineering a quaternary ammonium‐cleavable linker to conjugate diABZI STING agonist 3 (dSA3) with the HER2‐targeting antibody Trastuzumab. The optimized ISAC (TZ‐dSA3‐12) demonstrated high potency, stability, enhanced solubility, and reduced off‐target toxicity. The data showed that TZ‐dSA3‐12 potently activates the STING pathway in the tumor microenvironment through the synergistic action of the Fab and Fc regions of antibodies ( activity switch‐on ). In contrast, TZ‐dSA3‐12 exhibited ≈75 fold lower activity than dSA3 in normal immune cells, where activation relies solely on the Fc region without Fab‐mediated tumor antigen binding ( activity switch‐off ). Furthermore, systemic administration of TZ‐dSA3‐12 at a dose (1 mg kg −1 ) elicited robust and sustained antitumor effect in a manner dependent on the activation of innate immunity and adaptive immunity, including macrophages, dendritic cells (DCs) and CD8 + T cells, while minimizing systemic cytokine release. Notably, TZ‐dSA3‐12 also induced immunological memory to combat the growth of rechallenged tumors. This innovative quaternary ammonium‐linked STING agonist‐ISAC represents a promising avenue for the future development of STING‐targeted immunotherapy.

Lysosome-targeting chimeras (LYTACs) represent a revolutionary targeted protein degradation technology. However, the advancement of LYTACs faces substantial challenges due to the limited diversity of lysosome-trafficking receptors. In this study, we identified folate receptor α (FRα) as a new class of lysosome-trafficking receptors capable of facilitating the degradation of membrane proteins. Leveraging a polyvalent crosslinking strategy, we developed FRα-targeting chimeras (FRTACs), including epidermal growth factor receptor-targeting FR-Ctx and PD-L1-targeting FR-Atz. The optimized FRTACs demonstrated subnanomolar potency in eliminating cell-surface targets, with efficacy dependent on both FRα expression and lysosomal activity. Specifically, FR-Ctx inhibited cancer cell proliferation, while FR-Atz enhanced T cell-mediated cytotoxicity against tumor cells. FR-Atz exhibited robust PD-L1 degradation efficiency in vivo and elicited tumor-specific immune responses by reprogramming the tumor microenvironment from an immunosuppressive to an immunostimulatory state in both RM-1 and humanized B16F10 mouse models. These findings establish FRTACs as a promising platform for the design of tumor-targeting LYTACs.

The transcription factor Pax5 activates genes essential for B-cell development and function. However, the regulation of Pax5 expression remains elusive. The adaptor Rack1 can interact with multiple transcription factors and modulate their activation and/or stability. However, its role in the transcriptional control of B-cell fates is largely unknown. Here, we show that CD19-driven Rack1 deficiency leads to pro-B accumulation and a simultaneous reduction in B cells at later developmental stages. The generation of bone marrow chimeras indicates a cell-intrinsic role of Rack1 in B-cell homeostasis. Moreover, Rack1 augments BCR and TLR signaling in mature B cells. On the basis of the aberrant expression of Pax5-regulated genes, including CD19, upon Rack1 deficiency, further exploration revealed that Rack1 maintains the protein level of Pax5 through direct interaction and consequently prevents Pax5 ubiquitination. Accordingly, Mb1-driven Rack1 deficiency almost completely blocks B-cell development at the pro-B-cell stage. Ectopic expression of Pax5 in Rack1-deficient pro-B cells partially rescues B-cell development. Thus, Rack1 regulates B-cell development and function through, at least partially, binding to and stabilizing Pax5.

Thirty-one new 10,12-disubstituted aloperine derivatives were subtly constructed through a selective oxidation on the 10-α-C–H induced by sulfonyl and a nucleophilic substitution with the stereoselectivity and scalability. Of them, compound 6b displayed a moderate anti-human coronavirus OC43 (HCoV-OC43) potency and blocked the viral entry stage through a host mechanism of action. Using chemoproteomic techniques, both transmembrane serine protease 2 (TMPRSS2) and scavenger receptor class B type 1 (SR-B1) proteins, which act as host cofactors of viral entry, were identified to be the direct targets of 6b against HCoV-OC43. Furthermore, 6b may deactivate the TMPRSS2 by inducing a change in protein conformation, rather than binding to its catalytic center, thus suppressing the viral membrane fusion. Accordingly, our study provided key scientific data for the development of aloperine derivatives into a new class of antiviral candidates against human β-coronavirus, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Immune checkpoint therapies (ICT) have achieved unprecedented efficacy in multiple cancer treatments, but are still limited by low clinical response rates. Identification of immunogenic cell death (ICD)-inducing drugs that can induce tumor cell immunogenicity and reprogram the tumor microenvironment is an attractive approach to enhance antitumor immunity. In the present study, Raddeanin A (RA), an oleanane class triterpenoid saponin isolated from Anemone raddeana Regel, is uncovered as a potent ICD inducer through an ICD reporter assay combined with a T cell activation assay. RA significantly increases high-mobility group box 1 release in tumor cells and promotes dendritic cell (DC) maturation and CD8+ T cell activation for tumor control. Mechanistically, RA directly binds to transactive responsive DNA-binding protein 43 (TDP-43) and induces TDP-43 localization to mitochondria and mtDNA leakage, leading to cyclic GMP-AMP synthase/stimulator of interferon gene-dependent upregulation of nuclear factor κB and type I interferon signaling, thereby potentiating the DC-mediated antigen cross-presentation and T cell activation. Moreover, combining RA with anti-programmed death 1 antibody effectively enhances the efficacy of ICT in animals. These findings highlight the importance of TDP-43 in ICD drug-induced antitumor immunity and reveal a potential chemo-immunotherapeutic role of RA in enhancing the efficacy of cancer immunotherapy.

Hand, foot, and mouth disease (HFMD) caused by enterovirus A71 (EV-A71) infection, currently lacks specific preventive and therapeutic interventions. Here, we demonstrated that Pien Tze Huang (PZH) could dose-dependently inhibit EV-A71 replication at the cellular level, resulting in significant reductions in EV-A71 virus protein 1 (VP1) expression and viral yields in Vero and human rhabdomyosarcoma cells. More importantly, we confirmed that PZH could protect mice from EV-A71 infection for the first time, with Ribavirin serving as a positive control. PZH treatment reduced EV-A71 VP1 protein expression, viral yields in infected muscles, and improved muscle pathology. Additionally, we conducted a preliminary mechanism study using quantitative proteomics. The results suggested that the suppression of the PI3K/AKT/mTOR and NF-κB signaling pathways may contribute to the anti-EV-A71 activity of PZH. These findings provide strong evidence supporting the potential therapeutic application of PZH for EV-A71 infection management.

Using chemoproteomic techniques, we first identified EIF2AK2, eEF1A1, PRDX3 and VPS4B as direct targets of berberine (BBR) for its synergistically anti-inflammatory effects. Of them, BBR has the strongest affinity with EIF2AK2 via two ionic bonds, and regulates several key inflammatory pathways through EIF2AK2, indicating the dominant role of EIF2AK2. Also, BBR could subtly inhibit the dimerization of EIF2AK2, rather than its enzyme activity, to selectively modulate its downstream pathways including JNK, NF-κB, AKT and NLRP3, with an advantage of good safety profile. In EIF2AK2 gene knockdown mice, the inhibitory IL-1β, IL-6, IL-18 and TNF-α secretion of BBR was obviously attenuated, confirming an EIF2AK2-dependent anti-inflammatory efficacy. The results highlight the BBR's network mechanism on anti-inflammatory effects in which EIF2AK2 is a key target, and inhibition of EIF2AK2 dimerization has a potential to be a therapeutic strategy against inflammation-related disorders.

With the development of small-molecule immunotherapy drugs, its combination with the programmed cell death ligand 1/programmed cell death protein 1（PD-L1/PD-1） antibodies would provide a new opportunity for cancer treatment. Therefore, targeting PD-L1/PD-1 axis by small-molecule drug is an attractive approach to enhance antitumor immunity and considered as the next generation of tumor immunotherapy. In the present study, we investigated the anti-tumor role of salvianolic acid B（SAB） by regulating the PD-L1 level in tumors. Changes of total PD-L1 and membrane PD-L1 levels were determined by Western blot, flow cytometry and PD-1/PD-L1 interaction assays. The expression of mRNA level of PD-L1 was detected by real-time PCR. The cytotoxicity of activated peripheral blood mononuclear cell（PBMC） cells toward co-cultured tumor cells was measured by cell impedance assay and crystal violet experiment. Surface plasma resonance technique was used to analyze the direct interaction between SAB and ubiquitin carboxyl-terminal hydrolase 2（USP2）. The antitumor effect of SAB in vivo was examined by C57BL/6 mice bearing MC38 xenograft tumor（all animal experiments were conducted in accordance with the Animal Ethics Committee of the Institute of Medicinal Biotechnology,Chinese Academy of Medical Sciences）. Western blot and flow cytometry assay showed that SAB can significantly downregulate the abundance of PD-L1 in RKO and PC3 cells in dose-and time-dependent manner. PD-1/PD-L1binding assay revealed that SAB reduces the binding of tumor cells to recombinant PD-1 protein. Mechanism studies revealed that SAB can bind directly to USP2 protein and inhibit its activity, thus promote the ubiquitinproteasome pathway degradation of PD-L1 proteins. In addition, Cell impedance and crystal violet staining indicated that SAB enhances the killing activity of co-cultured PBMC cells toward tumor cells. MC38 tumor transplanted mouse experiments revealed that SAB treatment displayed significant suppression in the growth of MC38 tumor xenografts in C57BL/6 mice with an inhibition rate of 63.2% at 20 mg·kg-1. Our results demonstrate that SAB exerts its anti-tumor activity by direct binding and inhibiting the activity of USP2 and reducing the PD-L1 level. Our study provides an important material basis and scientific basis for the potential application of SAB in tumor immunotherapy drug targeting USP2-PD-L1 axis.

Seven photoaffinity-based and sixteen biotin-based berberine (BBR) probes were constructed and screened for their effects on c-Jun N-terminal protein kinases (JNK) phosphorylation (p-JNK) suppression at the cellular level. Taking active-photoaffinity probe 7c as a chemical tool, we first identified mitogen-activated protein kinase 7 (MAP2K7), an upstream protein on the JNK/stress activated protein kinase (SAPK) pathway, as a direct proteomic target of BBR using activity-based protein profiling (ABPP) and other chemical proteomic techniques. Furthermore, BBR's inhibitory effect on p-JNK was significantly attenuated in both the MAP2K7-knockdown and models, indicating a MAP2K7-dependent inhibition on the JNK signaling pathway. For the first time, we demonstrate the unique mechanism of BBR that directly targets MAP2K7 to inhibit p-JNK rather than JNK activity with the advantages of multiple activities and a good safety profile. [GRAPHICS] .

Sixty-one palmatine (PMT) derivatives, of which twenty-eight were new, were synthesized and evaluated for their anti-fibrogenic activities via collagen type I α 1 (COL1A1)-promoter based luciferase model in LX-2 cells, taking 2,3,10-trimethoxy-9-p-isopropyloxyprotopalmatine bromide (1) as the lead. Among them, compound 3a exerted the highest potency with the IC50 value of 8.19 μmol/L and SI value of 8.59, and reduced the expressions of multiple fibrogenic biomarkers, including COL1A1, TGF-β1, α-SMA and TIMP1 in a dose-dependent manner. In addition, it significantly reduced liver steatosis and inflammation, and especially attenuated the degree of liver fibrosis in choline-deficient, l-amino acid-defined, high-fat diet (CDAHFD)-induced NASH mice model in vivo. Mechanism study indicated that it significantly ameliorated liver injury by activating farnesoid X receptor (FXR). BDL-induced fibrosis rats model further verified its liver-protective and anti-fibrosis activities. Therefore, PMT derivatives constituted a new family of non-steroidal FXR agonists as anti-NASH candidates, with the advantage of good safety profile, and are worthy for further investigation.