OSW-1, a steroidal disaccharide isolated from the bulbs of Ornithogalum saundersiae, has been extensively studied for its extremely potent cytotoxicity against the National Cancer Institute's 60 cancer cell lines with an average IC50 of 0.78 nM, while exhibiting selectivity toward normal cells. Although OSBP and ORP4L have been identified as its binding targets, their known functions appear insufficient to account for the compound's exceptional potency, suggesting the involvement of additional mechanisms and targets. Therefore, elucidating novel target proteins associated with its activity is essential for the further development of this molecule. Here, we disclose that OSW-1 can block the glycolytic pathway and trigger compensatory mitochondrial oxidative phosphorylation. This previously uncharacterized mechanism is relevant to the key rate-limiting enzyme, enolase 1 (ENO1), which shows subnanomolar affinity with OSW-1. Our study repurposes OSW-1 to be a small-molecule probe to investigate the function of ENO1 and a promising candidate for metabolism-targeted anticancer therapy.

BACKGROUND:Integrin β3 orchestrates thrombotic and inflammatory processes through bidirectional signaling, with Src acting as a key downstream effector. OBJECTIVES:To dissect this axis, we generated SrcE97A mice that selectively disrupt the interaction between Src and integrin β3, impairing outside-in signaling while preserving inside-out signaling through integrin β3. METHODS:Platelet function was assessed through clot retraction, irreversible aggregation, thrombus stabilization under flow, and soluble fibrinogen binding. Thrombosis models included FeCl3-induced carotid artery and laser-induced cremaster arteriole injury for arterial thrombosis, and the inferior vena cava ligation for deep vein thrombosis. Hemostasis was evaluated in parallel. The impact on cerebral infarction was measured in vivo. In addition, the biological functions of αvβ3 outside-in signaling were also analyzed. RESULTS:The SrcE97A mutation impaired outside-in signaling while preserving inside-out signaling in platelets. This suppressed αIIbβ3-mediated clot retraction, secondary aggregation, and thrombus stabilization under flow. SrcE97A mice showed reduced arterial and venous thrombosis without hemostatic compromise. These antithrombotic effects were reinforced by protection against cerebral infarction in vivo. Furthermore, the E97A mutation influenced immunologic regulation. Specifically, it promoted pulmonary inflammation by skewing T helper (Th) cell differentiation toward Th1, via disruption of the αvβ3-Src-STAT4/6 signaling axis. CONCLUSIONS:These findings establish integrin β3 as a dual regulator of thrombosis and immune homeostasis. Selective disruption of outside-in signaling attenuates pathologic thrombosis without compromising hemostasis and reshapes Th1/Th2 balance. Targeting the Src E97 interface may offer strategies to inhibit pathologic thrombosis and modulate inflammation.

BACKGROUND:Given the persistently high morbidity and mortality of heart failure (HF), targeting myocardial remodeling, particularly pathological hypertrophy and fibrosis, has become a major therapeutic priority. RhoA (Ras homolog gene family member A), a small GTPase governing cytoskeletal reorganization and cell migration, plays a pivotal role in this process. However, RhoA has long been considered undruggable because of its high-affinity binding to GDP/GTP and the absence of well-defined druggable pockets. METHODS:Structural analyses comparing RhoA-GTP and RhoA-GDP conformations, combined with surface plasmon resonance-based screening, were used to identify a RhoA inhibitor. The underlying mechanism was validated in cultured cells and 3-dimensional myocardial tissue models. Therapeutic efficacy was assessed across multiple species of HF models and supported by multiomics analyses linking RhoA activation to human HF. Key findings were further confirmed by multiplex immunohistochemistry and pulldown assays in human heart specimens. RESULTS:We identified an unrecognized cryptic pocket adjacent to GDP in RhoA. A natural product, AH001, selectively occupied this pocket and interacted with GDP, thereby stabilizing the interaction between RhoA and its endogenous inhibitor, RhoGDIα (Rho GDP-dissociation inhibitor 1). AH001 suppressed downstream signaling by reducing MRTFA (myocardin-related transcription factor A) nuclear translocation and downregulating fibrosis- and hypertrophy-related proteins. Moreover, AH001 disrupted pathological crosstalk between Mrtfa+ cardiomyocytes and fibroblasts. Consequently, AH001 markedly attenuated myocardial remodeling in multiple HF animal models, as well as in 3-dimensional myocardial tissue models. CONCLUSIONS:These findings establish pharmacological inhibition of RhoA activation as a viable strategy to mitigate myocardial remodeling in HF and provide a conceptual framework for developing reversible inhibitors against previously undruggable small GTPases.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a common metabolic disorder characterized by excessive lipid accumulation in the liver, insulin resistance, and impaired glucose metabolism. Empagliflozin (EGZ) effectively reduces liver fat, enhances insulin sensitivity, and improves glucose metabolism in various metabolic conditions. To specifically target hepatocytes, glycyrrhetinic acid (GA) was incorporated into lipid nanoparticles (LNPs), creating a GA-targeted EGZ@LNP-GA drug delivery system designed to enhance the therapeutic effects of EGZ on MASLD. The EGZ@LNP-GA formulation was thoroughly characterized by evaluating particle size, encapsulation efficiency, and in vitro release kinetics. In vitro studies showed that EGZ@LNP-GA exhibited high specificity for LO2 cells compared to HeLa and HUVEC cells. Treating LO2 cells with EGZ@LNP-GA diminished lipid accumulation, as demonstrated by Oil Red O staining and triglyceride (TG) content assays. Western blot analysis revealed that EGZ@LNP-GA downregulated lipogenic factors SREBP1c and FAS, while upregulating the lipid oxidation regulator SIRT1, indicating its potential to enhance lipid metabolism. In an HFD-induced steatosis model, EGZ@LNP-GA reduced hepatic lipid accumulation, lowered inflammatory cytokines, improved insulin sensitivity, and enhanced glucose metabolism. These findings support the GA-targeted EGZ@LNP-GA system as a promising strategy for MASLD-related metabolic dysfunction.

Ubiquitin-specific protease 7 (USP7) is a key member of the deubiquitinating enzyme family. It is abnormally overexpressed in various malignancies, including breast cancer, chronic lymphocytic leukemia, and prostate cancer. By regulating pathways such as the p53-MDM2 signaling axis, USP7 promotes tumorigenesis and progression, making it a highly promising therapeutic target for anticancer treatment. Although multiple USP7 inhibitors have been reported, existing screening and evaluation assays exhibit limitations: the ubiquitin-phospholipase A2 (Ub-PLA2) assay frequently produces false-positive results, while the ubiquitin-rhodamine (Ub-Rho) assay is susceptible to interference from compound autofluorescence. To address this challenge, we developed a fluorescence polarization (FP) assay. This employs a rationally designed strategy that exhibits excellent characteristics, making it a simple-to-operate and cost-effective method, suitable for the evaluation of compound bioactivity against USP7. To further validate the practicality and reliability of this FP assay, we conducted a structure-based drug design campaign involving two rounds of systematic structural optimization, yielding 51 novel derivatives featuring pyrazolo[4,3-d]pyrimidine and piperidol scaffolds. Following FP evaluation and Ub-Rho enzyme activity validation, we performed a comprehensive structure-activity relationship (SAR) analysis. Ultimately, in vitro cellular assays identified three compounds (LC-U7-44, LC-U7-48, and LC-U7-50) that exhibit potent USP7 inhibitory activity alongside favorable cellular anti-proliferative effects. Overall, the established FP assay in this study closes a methodological gap in the evaluation of USP7 inhibitors, and the detailed SAR analysis provides a foundation for the further development of potent USP7 inhibitors.

Protein arginine methyltransferase 1 (PRMT1) plays a critical role in cancer, yet current PRMT1 modulators lack selectivity and rely on enzymatic inhibition. Here, we developed first-in-class PRMT1-targeting PROTAC degrader compound 4, designed based on the pharmacophore of our previously developed PRMT1 inhibitor. Compound 4 potently induces PRMT1 degradation in a concentration-, time-, and proteasome-dependent manner and exhibits high selectivity, with no detectable degradation of other common CRBN substrates and other type I PRMTs. It also effectively inhibited the growth of multiple cancer cell lines and exhibited a favorable pharmacokinetic profile. Molecular modeling suggests that the unique conformation of the PRMT1 dimerization arm promotes productive ternary complex formation with CRBN, providing a structural basis for selective PRMT1 degradation. Overall, this study demonstrates that compound 4 is a first-in-class PRMT1-targeting PROTAC degrader and highlights its value as a chemical tool for studying PRMT1 biology and its therapeutic potential in PRMT1-dependent cancers.

Peroxiredoxin 1 (PRDX1) is a pivotal antioxidant enzyme maintaining intracellular reactive oxygen species (ROS) balance. Deficiency of PRDX1 aggravates oxidative stress-related pathologies, whereas enhanced PRDX1 activity confers cytoprotection. Small-molecule agonists boosting PRDX1 peroxidase activity hold therapeutic promise, yet to date only two such agonists-rosmarinic acid (RA) and salvianolic acid B (SAB)-have been reported, both by our laboratory. These polyphenolic compounds are chemically rigid and recalcitrant to modification. Here, we resolved the crystal structure of PRDX1 in complex with salvianolic acid C (SAC), revealing a conserved danshensu substructure shared by SAC, RA, and SAB. Guided by this pharmacophore, we designed a scaffold hopping core structure and generated 160 derivatives via in situ click reaction. Among them, LC-PDA-01, a non-polyphenolic scaffold, exhibited the highest PRDX1 activation (EC50 = 111.8 nM). This work discloses the first structurally tractable PRDX1 agonist and highlights combinatorial click chemistry’s utility in transforming natural product motifs into drug-like molecules.

Metal ions are indispensable cofactors governing protein conformations and biofunctions. However, given the highly dynamic and subtle nature of metal ion modulations, capturing the metal-induced conformational dynamics and resolving these structural alteration details remain challenging using traditional structural biology methods. Herein, we apply native mass spectrometry (nMS) and 193-nm ultraviolet photodissociation (UVPD) to characterize the metal ion-dependent conformations of Kirsten rat sarcoma (KRAS). Achieving high sequence coverage, we established a biophysical baseline for the native Mg2+ cofactor, demonstrating how its structural impact modulates functional switch loops to stabilize a compact and inactive state of KRAS. Furthermore, we find the competitive substitution of Mg2+ by Zn2+ preserves the global compact topology but alters local stability, imparting the active site and switch regions with improved flexibility. Our work highlights how metal ion modulates on KRAS conformational dynamics, providing a sensitive MS-based analytical strategy for exploring the allosteric modulations across broader metal-proteins.

Abstract Molecular glue degraders (MGDs) offer a sophisticated, proximity-based approach to protein modulation. In this study, we introduce LJY-3-60, a novel proximity-inducing agent that unexpectedly triggers the potent and selective autodegradation of CRBN. Evidence from CRISPR-Cas9 screening and IP-MS reveals that this degradation process is strictly governed by the intrinsic CRL4 CRBN machinery, independent of any extrinsic E3 recruitment. Through a combination of cellular and biophysical characterizations, we demonstrate that LJY-3-60 acts as a molecular bridge to template CRBN homodimerization. This mechanism is unequivocally elucidated by the atomic-resolution co-crystal structure of the CRBN Midi -LJY-3-60 complex. The structure explicitly delineates the homodimerization interface, revealing how the ligand reorganizes the protein surface to stabilize a non-canonical architecture that drives trans-autoubiquitination and subsequent proteasomal degradation. Furthermore, LJY-3-60 serves as a highly effective, controllable off-switch to mitigate PROTAC-induced toxicity. Ultimately, this work delivers a robust chemical tool for modulating CRBN stability. By demonstrating how a small molecule can functionally mimic an endogenous E3 substrate’s degron to catalyse targeted autodegradation, this study establishes a rational structural framework for designing the next generation of self-destructive modulators in targeted protein degradation (TPD) therapeutics.

Hyperproteinemia is characterized by an unusually high plasma protein concentration (PPC). It affects both humans and animals. In this study, we used a silkworm model of hyperproteinemia to explore the mechanisms by which high PPC impairs female reproduction. Analysis of ovarian transcriptomes and qRT-PCR revealed that high PPC reduced the expression of vitellogenin (Vg) in the fat body and vitellogenin receptor (VgR) in the ovary. Further biochemical and gene expression analyses showed that high PPC decreased the titer of 20-hydroxyecdysone (20E) and the expression of key genes in the 20E-Vg/VgR signaling pathway. Exogenous 20E supplementation restored the expression of pathway components and effectively rescued female reproductive function. These findings offer new evidence for how high PPC affects female reproduction and provide a useful reference for future clinical research.

Transcriptional enhanced associated domain (TEAD) proteins, activated by YAP/TAZ, are oncogenic drivers. While parallel synthesis has advanced lead discovery, most libraries are assembled randomly or in a scaffold-centric manner, which results in low efficiency for inhibitor discovery. In this study, we leveraged a strategy integrating structure-based design with CuAAC-enabled parallel synthesis to build a covalent, hydrophobic-fragment library. Combined with in situ screening, this approach rapidly identified hits at 8.33%, among which LC-TEAD01 emerged as a selective inhibitor showing a 17-fold preference for NF2-deficient NCI-H226 cells. Biochemical and structural studies confirmed covalent engagement of the conserved cysteine and occupancy of the hydrophobic channel, disrupting YAP-TEAD interaction and suppressing TEAD-dependent transcription. In vivo, LC-TEAD01 inhibited tumor growth in NF2-deficient xenografts. Collectively, this work integrates structure-based design with CuAAC-enabled parallel synthesis and in situ screening, enabling rapid discovery of TEAD inhibitors and offering a generalizable route for targets with structurally defined pockets.

Abstract T cell engager (TCE) antibodies have been proven to be effective for a variety of hematological malignancies, with at least 10 marketed drugs to date; however, their efficacy in many solid tumors is still very limited due to a number of challenges. One of the leading challenges is the lack of tumor-specific targets, leading to an increased risk of on-target, off-tumor toxicity. An approach to address this challenge is to build a “prodrug” TCE that is conditionally activated in the tumor microenvironment (TME), taking advantages of various masking technology platforms that block the release of active TCE drug outside of the TME. To develop such a novel TCE molecule with improved tumor selectivity, potency, and safety profile, we first discovered a masking peptide specific to a CD3 VHH antibody that we previously developed, and demonstrated that the masked CD3 VHH can effectively reduce T cell activation several hundred-fold. Next, we fused this masking peptide to the CD3 VHH arm via a proteolytically-labile linker containing sequence motifs sensitive to proteases enriched in the TME, and showed that upon cleavage by proteases, the T cell-activating potency of such a masked CD3 VHH can be effectively recovered. Finally, with such a CD3 VHH armed with cleavable linker and masking peptide, we built a series of prodrug TCE molecules targeting ENPP3 (ectonucleotide pyrophosphatase/phosphodiesterase family member 3), a tumor associated antigen with good tumor selectivity for renal cell carcinoma. Our in vitro validation studies demonstrated that the potency of these TCE prodrugs in killing ENPP3+ tumor cells can be restored to a similar level as the unmasked, active TCE molecule upon proteolytic cleavage. Our in vivo studies using an ENPP3+ CDX model with transplanted human PBMCs demonstrated two important points: (i) the prodrug/masked ENPP3-targeted TCE can potently inhibit tumor growth to a comparable level as the unmasked version and clinical benchmarks, and (ii) the prodrug TCE can be dosed at least 100-fold higher than the active drugs without any observed toxicity, suggesting a greatly improved safety profile. Further preclinical development of these ENPP3-targeted prodrug TCE molecules, including pharmacokinetics and toxicity studies in non-human primates, is ongoing. Citation Format: Li Chen, Lindi Wang, Yingyu Li, Xin Wang, Yang Xin, Mingzhu Shao, Jiangtao Ning, Ziyu Chen, Cheng Luo, Jingjing Nie, Yue Wu, Tingting Yang, Hai Huang, Miaomiao Song, Yu Liang. The discovery and development of a bispecific T cell engager prodrug targeting ENPP3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4410.

ABSTRACT Natural products, owing to their unique biological activities, possess the ability to interact with specific target proteins or regulatory networks, representing a valuable source of innovative drug candidates. However, target identification remains a major bottleneck in natural product‐based drug discovery, largely because of the chemical complexity of natural products and the heterogeneity of biological systems. To address these challenges, various complementary strategies have been developed, including experimental strategies such as chemical proteomics, and computational methods such as artificial intelligence‐driven methods. Nevertheless, reliably advancing a candidate protein hit to a therapeutically relevant and physiologically validated target remains a critical challenge. Focusing on technologies for natural product target discovery, this review systematically summarizes the principles, methodologies, and practical applications of current approaches. Through representative case studies, we further propose a reusable integrated experimental–computational workflow and illustrates how key targets and their modes of action can be identified in real‐world research scenarios. In addition, we discuss common technical and conceptual bottlenecks encountered during target discovery and proposes potential countermeasures. The review provides an actionable reference framework for natural product target identification, with the goal of reducing false‐positive findings and fragmented evidence, thereby improving the robustness of mechanism‐oriented studies and facilitating subsequent translational research.

Metabolic reprogramming and immune regulation are tightly interconnected processes that critically influence cancer progression. The efficacy of immunotherapy is limited in triple-negative breast cancer (TNBC) by metabolic abnormality and immunosuppressive microenvironment. However, the molecular mechanisms through which these alterations cooperate to drive immune evasion and tumor progression in TNBC remain poorly defined. Through transcriptomic profiling, we identified glycolysis and chemokine signaling as the dominant intersecting metabolic and immune pathways that distinguish TNBC from non-TNBC subtypes. Phosphoglycerate kinase 1 (PGK1) is associated with poor prognosis and with myeloid-derived suppressor cells (MDSCs), acting as a key metabolic node linking metabolic regulation to immune modulation. Functionally, PGK1 knockdown inhibited tumor growth in vitro and in vivo and reduced MDSC recruitment. Notably, PGK1 knockdown exerted a more pronounced antitumor effect under immune surveillance, accompanied by decreased infiltration of both monocytic and polymorphonuclear MDSCs and recovered CD8+ T cell function. Mechanistically, PGK1 increased lactate production and global lysine lactylation. Notably, histone H3 lysine 18 lactylation (H3K18la) at the CCL5 promoter served as a dominant and required epigenetic modification for PGK1-driven CCL5 transcription, thereby driving CCL5-dependent MDSC recruitment. P300 and class I HDACs were identified as candidate "writer" and "eraser" enzymes for PGK1-dependent H3K18la modification. Notably, combining the PGK1 inhibitor ABT-E79 with anti-PD-1 therapy synergistically decreased MDSC infiltration, recovered CD8+ T cell function, and elicited superior antitumor responses compared to monotherapy. Collectively, this study shows a mechanistic link between metabolic reprogramming and immune evasion, offering new therapeutic insights for TNBC. Starting from analyzing metabolic and immunological signaling pathways in cancers lacking clear therapeutic targets and treatment options, we aimed to identify metabolism-associated regulators of immune responses as potential therapeutic targets. Focusing on triple-negative breast cancer (TNBC), through transcriptomic profiling, we identified glycolysis and chemokine signaling as the dominant intersecting metabolic and immune pathways that distinguish TNBC from non-TNBC subtypes. Functional assays demonstrated that PGK1-driven glycolysis in TNBC cells promotes lactate accumulation and H3K18la, which subsequently induce transcriptional activation of CCL5 and recruitment of MDSCs, thereby impairing CD8+ T cell function and fostering an immunosuppressive tumor microenvironment that facilitates immune evasion. Furthermore, treatment with ABT-E79, a PGK1 inhibitor, enhances the antitumor immune efficacy of anti-PD-1 therapy.

The classical sequential workflow-individual compound screening, followed by stepwise design-synthesis-purification of derivatives-makes lead compound discovery time-consuming and cost-prohibitive. Herein, we developed Affinity Selection Thin-Layer Chromatography (AS-TLC) as a method for high-throughput screening (HTS), enabling efficient and cost-effective screening of compound mixtures. Furthermore, we integrated activity assays with AS-TLC to minimize target protein consumption and lower the false positive rate, through which we identified a YTH domain-containing protein 1 (YTHDC1) inhibitor, fragment YD, with a half-maximal inhibitory concentration (IC50) of 20.01 ± 3.30 µm. Building on this hit, we applied copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry for rapid structural modification and compound library construction, ultimately obtaining LC-YD03 with significantly enhanced inhibitory activity (IC50 = 41.98 ± 6.24 nm). The interaction mode between LC-YD03 and YTHDC1 was further clarified via X-ray crystallographic analysis. This study demonstrates that the AS-TLC-activity assay platform enables efficient hit screening from compound mixtures; subsequent click chemistry-based combinatorial library technology accelerates lead compound optimization. Collectively, this workflow significantly reduces the time and cost associated with lead compound discovery.