(a) Western blot analysis of glycoprotein patterns in MDA-MB-231 cells after treatment with the indicated concentrations of penfluridol for 24 hours. Red arrowhead, immature glycoproteins. (b) Western blot analysis of protein expression pattern of ACE2 in HEK293T cells after treatment with the indicated concentrations of penfluridol for 24 hours. (c) In HEK293T cells, FLAG-tagged ACE2 was treated for 24 hours, collected by immunoprecipitation, subjected to SDS-PAGE, and analyzed by western blot. (d) Pearson correlation coefficient of three independent groups of (c) through N-glycan profiling.

Target-based drug screening typically relies on biochemical or affinity-based assays to identify compounds that modulate or bind to purified target proteins in vitro. However, additional cellular validation is essential to confirm genuine drug-target engagements. Integrating screening and validation within a single cellular assay could greatly expedite the drug discovery process. Herein, we developed a cellular ligand discovery method called CPSEA (cellular protein stability enhancement assay), which leverages the biophysical principle of ligand-induced stabilization of target proteins containing destabilizing mutations. Using CPSEA, we identified arteannuin B and colchicine as novel ligands for FKBP12 and KRASG12S, respectively. Importantly, we introduced both experimental and computational strategies to identify destabilizing mutations, thereby broadening the applicability of CPSEA for target proteins with and without known stabilizing ligands. Overall, CPSEA represents a powerful cell-based screening strategy with significant potential in target-based drug discovery.

Dysregulation of autophagy and mitophagy is a hallmark of neurodegenerative diseases, including Alzheimer's disease (AD). Chemical intervention targeting these pathways has emerged as one of the promising therapeutic strategies for neurodegenerative disorders. Here, we identified beauvericin as a candidate molecule that regulates autophagy and mitophagy through an organelle phenotypes-based high-throughput screening of a marine natural products library. Mechanistic analyses revealed that beauvericin engages NIPSNAP2 and promotes its activation, and enhances autophagic flux and mitophagy across multiple cell types. Moreover, in AD-relevant cellular models, beauvericin significantly reduced amyloid-β (Aβ) levels via lysosome-dependent degradation of BACE1. Collectively, these findings demonstrate that beauvericin activates autophagy and mitophagy via NIPSNAP2 and that chemical activation of these pathways can ameliorate AD-relevant cellular phenotypes, supporting its potential as a chemical intervention for neurodegenerative diseases.

The bidirectional crosstalk between the nervous system and tumors has emerged as a transformative new frontier in both precision oncotherapy and mechanism-driven cancer pain management. Marine natural products with inherent neuroactive properties exhibit unparalleled intervention advantages for targeting this complex pathophysiological axis. Herein, we systematically and prospectively dissect the multi-layered bidirectional communication between the nervous system and malignancies, comprehensively summarize the pivotal contributions of marine-derived bioactive molecules to advances in neuroscience and antitumor therapeutics, and finally provide an outlook on and a call for integrated, interdisciplinary collaboration to enable transformative breakthroughs in the development of marine neuropharmacological agents targeting the nerve–tumor crosstalk axis.

(a) qPCR analysis of the GRP78 and CHOP expression in MDA-MB-231 cells after the treatment with the indicated concentrations for 6 hours. (b) Western blot analysis of Integrin β1 expression pattern in MDA-MB-231 cells after indicated drugs treatment for 24 hours. (c) Western blot analysis of protein expression pattern of Integrin β1 in MDA-MB-231 cells treated with the indicated siRNA.

Abstract von Hippel–Lindau (VHL) is a tumor suppressor frequently inactivated in renal cell carcinoma (RCC), and its loss is associated with aberrant DNA methylation. Here we demonstrate that VHL-deficient RCC cells are highly vulnerable to DNA methyltransferase (DNMT) inhibitors. US Food and Drug Administration-approved DNMT inhibitors, such as decitabine and azacitidine, and investigational agents including RX-3117 and SGI-1027 selectively suppressed the growth of VHL-deficient RCC cells. Mechanistically, VHL loss leads to HIF-2α-dependent transcriptional upregulation of DNMT1, resulting in widespread CpG hypermethylation. Transcriptomic profiling and an RNA interference-based rescue screen identified KCNK3, a putative tumor suppressor, as a key mediator of DNMT inhibitor-induced synthetic lethality in VHL-deficient RCC. The KCNK3 promoter is hypermethylated and transcriptionally repressed in VHL-deficient RCC, where treatment with DNMT inhibitors reverses this methylation, restoring KCNK3 expression and resulting in cell growth inhibition. Silencing KCNK3 significantly attenuated the antitumor effects of DNMT inhibitors both in vitro and in vivo. Further mechanistic analysis showed that KCNK3 reactivation triggers TNF-α, MAPK and apoptotic signaling pathways, contributing to the observed synthetic lethality. Collectively, these findings establish DNMT inhibition as a synthetic lethal strategy in VHL-deficient RCC and highlight a potential therapeutic vulnerability for personalized treatment approaches.

Myelin debris and apoptotic cells generated after intracerebral hemorrhage (ICH) contribute to inflammatory responses and hinder recovery. Efficient clearance of cellular debris by microglia is indispensable for neurological recovery and functional restoration. In this study, we demonstrate that pyridoxal (PL), a form of vitamin B6, plays a pivotal role in enhancing microglial phagocytic activity. Using an ICH mouse model, we found that vitamin B6 deficiency increased the accumulation of demyelinated myelin basic protein (dMBP) and elevated cell death, whereas PL supplementation significantly reduced the dMBP-positive area and the burden of apoptotic cells, thereby improving neurological function. Furthermore, knockout of pyridoxal kinase (Pdxk) in oligodendrocytes improved neurological recovery after ICH. Mechanistically, Pdxk knockout in oligodendrocytes upregulated interleukin-33 (Il33) secretion, thereby promoting microglial phagocytosis. Blocking the Il33 receptor with neutralizing antibodies abolished the protective effects of Pdxk deletion, whereas administration of recombinant Il33 in ICH mice enhanced debris clearance and functional recovery. Collectively, our findings suggest that oligodendrocyte Pdxk may represent a key therapeutic target for enhancing recovery after ICH through Il33-mediated modulation of microglial function.

Immune checkpoint blockades have shown great potential in cancer therapy. However, achieving efficient recruitment and activation of T cells while blocking immune suppression remains a critical challenge. Current strategies mainly focus on the blockade of the PD-1/PD-L1 axis, with limited attention to reprogramming immune functions on the tumor cell surface. Here, we report a "localized oxidation-covalent assembly" strategy that achieves precise modification of PD-L1 on the cell surface through glycan oxidation, thereby harnessing bioorthogonal reactions to induce the in situ construction of artificial topological nanostructures (ATNs), which subsequently augment T cell-mediated antitumor immunity. ATNs not only block the PD-1/PD-L1 axis to relieve immune suppression but also recruit and activate T cells through transmembrane bridging interactions, mimicking bispecific T cell engagers (BiTEs) and markedly enhancing antitumor immune responses. Mechanistic studies revealed that N-glycosylation sites are critical for probe-mediated aldehyde modification of PD-L1. We further demonstrated that the ATNs achieve spatially precise T cell recruitment and activation via PD-L1-dependent localization, enabling programmable immune regulation. Overall, this approach not only underscores the potential of glycan oxidation-driven self-assembly in immune modulation but also provides a versatile chemical biology tool for the precise reprogramming of immune checkpoint functions.

Supplementary Figure S1-S4 Supplementary Table S1

Immune suppression within the tumor microenvironment remains a major barrier to effective immunotherapy, yet the molecular mechanisms that constrain IFN-I signaling in tumor cells are incompletely understood. PARP7, a mono-ADP-ribosyltransferase, has been reported to suppress IFN-I signaling by modifying TBK1. Here, we identify PARP7 as a previously unrecognized acetylation substrate of the histone acetyltransferases p300/CBP. We show that p300/CBP-mediated acetylation of PARP7 at lysine 32 markedly enhances its stability, thereby reinforcing repression of the TBK1-IRF3-STAT1 axis and suppressing IFN-?-induced expression of interferon-stimulated genes, including CXCL10. Beyond their established function as transcriptional co-activators that support tumor cell proliferation, our findings uncover a direct role for p300/CBP in shaping innate antitumor immunity. Pharmacological inhibition of p300/CBP catalytic activity by A-485 abolishes PARP7 acetylation, accelerates its proteasomal turnover, and restores IFN-I signaling. In immunocompetent tumor models, this is accompanied by enhanced CD8+ T-cell infiltration and effector function. Together, these findings define a p300/CBP-PARP7 regulatory axis, uncover a non-canonical role for p300/CBP in innate immune suppression, and nominate this pathway as a tractable target for restoring antitumor immunity.

Ring Finger Protein 4 (RNF4) recognizes poly-SUMOylated proteins via its SUMO-Interacting Motifs (SIMs) and subsequently ubiquitinates them, thus effecting some key regulatory proteins involved in cancer development and progression. Our previous study found RNF4 was a potential target for HCC interruption. However, none of its inhibitor has been developed so far. Targeting RNF4 for degradation, rather than mere binding, emerged as a promising alternative strategy. To this end, we designed, synthesized and evaluated 28 PROTACs targeting RNF4 based on a reported covalent binder. Among them, RD12 was identified as the lead compound through a systematic screening. RD12 showed efficient RNF4 degradation activity as well as potent anti-proliferative activity in multiple HCC cell lines. Notably, it exhibited significant anti-tumour activity in a HCC mouse model without noticeable side effects. Mechanistic studies confirmed RD12 degraded RNF4 via the ubiquitin-proteasome system, and it induced DNA damage and apoptosis. These findings collectively underscore RD12 as the first pioneering RNF4 degrader and indicate its potential for HCC therapy.

A catalyst-free visible-light-induced protocol for the C4-selective C-H arylation of pyrimidine derivatives has been developed using arylazo sulfones as aryl radical precursors. Under mild conditions and without external photocatalysts or transition metals, a broad range of arylated pyrimidines were obtained in moderate to excellent yields. Mechanistic studies, including radical trapping experiments and light-control tests, support a visible-light-triggered radical pathway. Selected compounds exhibited promising antiproliferative activities against A549 and MCF-7 cell lines.

Abstract The inability to selectively trigger therapeutic hypothermia independent of environmental cooling has hindered causal analysis of its broader physiological benefits. Here, we employed P57, a natural product that pharmacologically induces therapeutic hypothermia circumventing external cold stress, to identify the neuronal substrates underlying hypothermia-induced antitumor effects. Integrating functional ultrasound imaging, activity-dependent neuronal labeling, and single-nucleus RNA sequencing, we identify a previously unrecognized population of Foxp2 ⁺ neurons in the medial preoptic area (MPA) that are selectively activated by P57. Inhibition of MPA Foxp2 + neurons abolishes P57-induced hypothermia, whereas the paraventricular hypothalamus (PVH) serves as a critical downstream node. Importantly, targeted activation of MPA Foxp2 ⁺ neurons is sufficient to induce sustained hypothermia, suppress systemic metabolism, and inhibit tumor growth, providing causal evidence that neuronally driven reductions in core body temperature can exert antitumor effects. Together, these findings establish MPA Foxp2 ⁺ neurons as a controllable node for therapeutic hypothermia induction and demonstrate that neuromodulation of defined neuronal populations can achieve therapeutic benefit by directly controlling physiological states.

Background Identifying effective therapeutic drugs in the intricate tumor microenvironment (TME) is challenging, further complicated by the lack of a systematic framework for analyzing TME perturbations in response to therapeutic interventions.Methods To address this, we established the single-cell RNA sequencing repository of immunomodulatory drugs resource and used the L1000 platform for unbiased screening of 739 immune-modulating compounds across various cancers.Results Drug responses in mouse model revealed 12 distinct meta-programs associated with TME remodeling, enriched in biological processes such as antigen presentation, tissue repair, and salt stress response. Notably, myeloid-derived suppressor cells were markedly reduced in responsive TMEs compared with other cell types, underscoring their key immunosuppressive role. We developed an MP scoring algorithm to quantify TME responsiveness, which successfully identified allopurinol—a gout medication—as a potent enhancer of anti-programmed cell death protein-1 therapy. This combination led to significant tumor-free outcomes (4/6) in vivo.Conclusions This work provides a robust framework for assessing TME remodeling that uncovers genes and compounds that significantly modulate immunotherapeutic efficacy.

(a) The stability of proteins was determined by cellular thermal shift at different concentrations of penfluridol.

(a) Western blot analysis of protein expression pattern of PD-L1 in HEK293T cells after the exposure to the indicated concentrations of penfluridol for 24 hours. (b) Protein expression pattern of PD-L1 in B16-F10 cells after treatment with the indicated concentrations of penfluridol for 12 hours as determined by western blot. (c) The toxicity effect of penfluridol on B16-F10 (top) and B16-OVA (bottom) cells. (d) Western blot analysis of protein expression of MAN1A1 in B16-OVA cells treated with the indicated siRNA.

Natural products play a crucial role in new drug development, but their druggability is often limited by uncertain molecular targets and insufficient research on mechanisms of action. In this study, we developed a new RPL19-TRAPKI-seq method, combining CRISPR/Cas9 and TRAP technologies, to investigate these mechanisms. We identified and validated seven ribosomal large subunit surface proteins suitable for TRAP, selecting RPL19 for its high enrichment. We successfully established a stable cell line expressing EGFP-RPL19 using CRISPR knock-in and verified its efficiency and specificity in enriching ribosomes and translating mRNA. Integrated with next-generation sequencing, this method allows precise detection of translating mRNA. We validated RPL19-TRAPKI-seq by investigating rapamycin, an mTOR inhibitor, yielding results consistent with previous reports. This optimized TRAP technology provides an accurate representation of translating mRNA, closely reflecting protein expression levels. Furthermore, we investigated SBF-1, a 23-oxa-analog of natural saponin OSW-1 with significant anti-tumor activity but an unclear mechanism. Using RPL19-TRAPKI-seq, we found that SBF-1 exerts its cytotoxic effects on tumor cells by disturbing cellular oxidative phosphorylation. In conclusion, our method has been proven to be a promising tool that can reveal the mechanisms of small molecules with greater accuracy, setting the stage for future exploration of small molecules and advancing the fields of pharmacology and therapeutic development.

Molecular docking is a widely used technique in computer-aided drug design, facilitating the prediction of ligand-receptor interactions. However, selecting the effective docking program for a given ligand remains challenging due to the diversity of ligand and the varying algorithms employed by docking software. In this study, we investigated the relationship between several ligand physicochemical properties and the performance of various molecular docking programs. By integrating ligand physicochemical properties and molecular fingerprints, we constructed a multidimensional ligand attribute set to represent the ligands comprehensively. These attributes were utilized to develop a machine learning classifier capable of predicting the effective docking program for specific ligands. The classifier was validated through virtual screening against pyridoxal kinase (PDXK), leading to the identification of several potential PDXK inhibitors. Our results demonstrate the efficacy of combining machine learning with ligand multidimensional descriptors analysis to improve docking program selection, offering a novel approach to enhance the efficiency and accuracy of molecular docking in drug discovery.

Dear Editor, Chronic hepatitis B virus(HBV)is a global health problem closely associated with a spectrum of liver diseases.Current clinical treatment options for HBV infection are generally not curative,highlighting the need for the development of novel therapeutics.Sodium taurocholate cotransporting polypeptide(NTCP)was identified as a functional receptor for HBV entry,making it a promising therapeutic target for developing novel anti-HBV agents.Although considerable efforts have been made to develop small molecule inhibitors against NTCP,many of these compounds suffer from low inhibitory potency and lack of efficacy in vivo.1 Therefore,the development of novel NTCP inhibitors with high specificity and efficacy is of great importance.