Background Breast cancer remains a critical unmet clinical challenge due to its aggressive metastasis behavior and limited treatment options. Topoisomerase inhibitors are widely available clinically but fail to address the compensatory upregulation of alternative isoforms in metastatic breast cancer. Techonology We engineered redox-responsive, carrier-free nanoassemblies (POD-2S-CPT NPs, PSC NPs) by co-assembling camptothecin (CPT, Topo I inhibitor) and podophyllotoxin (POD, Topo II inhibitor) linked via a disulfide bond. This design achieved high drug loading and leveraged the tumor microenvironment (TME), specifically elevated glutathione (GSH), for targeted drug release and maximized the synergistic therapeutic effect of Topo I/II inhibition. Results The PSC NPs demonstrated GSH-responsive drug release and selective cytotoxicity against MCF-7 cells. Mechanistically, they induced necroptosis via concurrent nuclear Topo I/II inhibition, mitigating compensatory resistance. In vivo, PSC NPs exhibited potent antitumor efficacy and significantly suppressed lung metastasis, without significant systemic toxicity. RNA-seq analysis revealed concurrent downregulation of the CXCL1/5-S100A8/9 axis in treated tumors, suggesting potential immunomodulatory effects alongside direct DNA damage. This self-assembled, redox-responsive nanoplatform enabled synergistic dual Topo I/II inhibition within the TME. It demonstrated potent anti-tumor and anti-metastatic activity with a favorable safety profile, presenting a promising potential strategy for metastatic breast cancer therapy.

Oxidative stress-induced mitochondrial damage and NLRP3/Caspase-1/GSDMD-mediated pyroptosis are pivotal pathological mechanisms in inflammatory bowel disease (IBD), which are often exacerbated by gut microbial dysbiosis. In this study, we designed and fabricated a carrier-free GMR hydrogel via Mg2+-mediated self-assembly of natural products glycyrrhizic acid (GA) and rosmarinic acid (RA) for the treatment of IBD. In established models of ulcerative colitis (UC) and Crohn's disease (CD), GMR hydrogel exhibited remarkable protective and therapeutic effects. Its efficacy is achieved through a dual mechanism: protecting intestinal epithelial cells (IECs) via ROS elimination and mitochondrial preservation, and mitigating pyroptosis by modulating the NLRP3/Caspase-1/GSDMD pathway, which collectively led to reduced IL-1 beta and IL-18 levels. Consequently, GMR hydrogel elicited comprehensive therapeutic effects, including the restoration of intestinal barrier integrity via upregulation of tight junction proteins (ZO-1, occludin, and claudin-1) and the remodeling of gut microbiota to enhance diversity and short-chain fatty acids (SCFAs) production. These improvements collectively created an anti-inflammatory microenvironment that ameliorated IBD symptoms. Collectively, these findings demonstrate that the multi-component self-assembled GMR hydrogel targets the core pathological pathways of mitochondrial damage and pyroptosis in IBD to restore intestinal homeostasis, positioning this biocompatible platform as a highly promising therapeutic strategy.

Experimental studies on DNA transposable elements (TEs) have been limited in scale, leading to a lack of understanding of the factors influencing transposition activity, evolutionary dynamics, and application potential as genome engineering tools. We predicted 130 active DNA TEs from 102 metazoan genomes and evaluated their activity in human cells. We identified 40 active (integration-competent) TEs, surpassing the cumulative number (20) of TEs found previously. With this unified comparative data, we found that the Tc1/mariner superfamily exhibits elevated activity, potentially explaining their pervasive horizontal transfers. Further functional characterization of TEs revealed additional divergence in features such as insertion bias. Remarkably, in CAR-T therapy for hematological and solid tumors, Mariner2_AG (MAG), the most active DNA TE identified, largely outperformed two widely used vectors, the lentiviral vector and the TE-based vector SB100X. Overall, this study highlights the varied transposition features and evolutionary dynamics of DNA TEs and increases the TE toolbox diversity.

Tandem repeat proteins (TRPs) are widely distributed and bind to a wide variety of ligands. DNA-binding TRPs such as zinc finger (ZNF) and transcription activator-like effector (TALE) play important roles in biology and biotechnology. In this study, we first conducted an extensive analysis of TRPs in public databases, and found that the enormous diversity of TRPs is largely unexplored. We then focused our efforts on identifying novel TRPs possessing DNA-binding capabilities. We established a protein language model for DNA-binding protein prediction (PLM-DBPPred), and predicted a large number of DNA-binding TRPs. A subset was then selected for experimental screening, leading to the identification of 11 novel DNA-binding TRPs, with six showing sequence specificity. Notably, members of the STAR (Short TALE-like Repeat proteins) family can be programmed to target specific 9 bp DNA sequences with high affinity. Leveraging this property, we generated artificial transcription factors using reprogrammed STAR proteins and achieved targeted activation of endogenous gene sets. Furthermore, the members of novel families such as MOON (Marine Organism-Originated DNA binding protein) and pTERF (prokaryotic mTERF-like protein) exhibit unique features and distinct DNA-binding characteristics, revealing interesting biological clues. Our study expands the diversity of DNA-binding TRPs, and demonstrates that a systematic approach greatly enhances the discovery of new biological insights and tools.

T cell exhaustion presents a major challenge for the efficacy of both immune checkpoint inhibitors (ICBs) and chimeric antigen receptor T (CAR-T) cell immunotherapies. To address this issue, we generate hypofunctional CAR-T cells that imitate the exhaustion state. By screening a Food and Drug Administration (FDA)-approved small molecule library using this model, we identify miltefosine as a potent molecule that restores the impaired function of CAR-T cells in a PD-1/PD-L1-independent manner. Impressively, in the terminally exhausted state where PD-1 antibody treatment is ineffective, miltefosine still enhances CAR-T cell activity. Single-cell sequencing analysis reveals that miltefosine treatment significantly increases the population of effector cells. Mechanistically, miltefosine improves impaired glycolysis and oxidative phosphorylation in hypofunctional CAR-T cells. In both allogeneic and syngeneic tumor models, miltefosine effectively enhances the solid tumor clearance ability of CAR-T cells and T cells, demonstrating its potential as an effective immunotherapeutic drug.

Supplementary Figure S1. Generation and characterization of cell lines for high content screening and GLI-luciferase reporter assays. Supplementary Figure S2. A high content screening identifies compound candidates that inhibit GLI2 ciliary localization. Supplementary Figure S3. Additional prostaglandins were examined in the GLI-luciferase reporter assay using 3T3/GLI-luc/GLI2 cells. Supplementary Figure S4. Comparative examination of dose-dependent effects of the prostaglandins identified from high content screening on the GLI-luciferase reporter activity mediated by SMO-WT, SMO-D473H and SMO-W535L respectively. Supplementary Figure S5. Examination of prostaglandins against SHH-N/SAG-induced HH pathway activity. Supplementary Figure S6. A CRE-luciferase reporter assay was used to examine cAMP-PKA levels. Supplementary Figure S7. Generation and examination of EP4 knockout monoclonal cell lines. Supplementary Figure S8. Examination of the expression of EP receptors and the activity of PGE1 in DAOY cells and growth comparison of wild-type DAOY cells and GLI2-overexpressed DAOY cells. Supplementary Figure S9. Molecular analyses of Med-113FH and Med-314FH xenografts. Supplementary Figure S10. Examination of the activities of PGE1 metabolites. Supplementary Figure S11. Examination of additional clinical prostaglandins.

Gene-specific primers used in this paper.

Chimeric antigen receptor (CAR) T cell therapy has limited efficacy against solid tumors, and one major challenge is T cell exhaustion. To address this challenge, we performed a candidate gene screen using a hypofunction CAR-T cell model and found that depletion of basic leucine zipper ATF-like transcription factor (BATF) improved the antitumor performance of CAR-T cells. In different types of CAR-T cells and mouse OT-1 cells, loss of BATF endows T cells with improved resistance to exhaustion and superior tumor eradication efficacy. Mechanistically, we found that BATF binds to and up-regulates a subset of exhaustion-related genes in human CAR-T cells. BATF regulates the expression of genes involved in development of effector and memory T cells, and knocking out BATF shifts the population toward a more central memory subset. We demonstrate that BATF is a key factor limiting CAR-T cell function and that its depletion enhances the antitumor activity of CAR-T cells against solid tumors.

Abstract Aberrant activation of the Hedgehog (HH) signaling pathway underlines the initiation and progression of a multitude of cancers. The effectiveness of the leading drugs vismodegib (GDC-0449) and sonidegib (LDE225), both Smoothened (SMO) antagonists, is compromised by acquisition of mutations that alter pathway components, notably secondary mutations in SMO and amplification of GLI2, a transcriptional mediator at the end of the pathway. Pharmacologic blockade of GLI2 activity could ultimately overcome these diversified refractory mechanisms, which would also be effective in a broader spectrum of primary tumors than current SMO antagonists. To this end, we conducted a high-content screening directly analyzing the ciliary translocation of GLI2, a key event for GLI2 activation in HH signal transduction. Several prostaglandin compounds were shown to inhibit accumulation of GLI2 within the primary cilium (PC). In particular, prostaglandin E1 (PGE1), an FDA-approved drug, is a potent GLI2 antagonist that overcame resistance mechanisms of both SMO mutagenesis and GLI2 amplification. Consistent with a role in HH pathway regulation, EP4 receptor localized to the PC. Mechanistically, PGE1 inhibited HH signaling through the EP4 receptor, enhancing cAMP-PKA activity, which promoted phosphorylation and degradation of GLI2 via the ubiquitination pathway. PGE1 also effectively inhibited the growth of drug refractory human medulloblastoma xenografts. Together, these results identify PGE1 and other prostaglandins as potential templates for complementary therapeutic development to circumvent resistance to current generation SMO antagonists in use in the clinic. Significance: These findings show that PGE1 exhibits pan-inhibition against multiple drug refractory activities for Hedgehog-targeted therapies and elicits significant antitumor effects in xenograft models of drug refractory human medulloblastoma mimicking GLI2 amplification.

The lead compound TBA, 3β-Hydroxy-lup-20(29)-ene-28-oic acid-3, 5, 6-trimethylpyrazin-2-methyl ester, which exhibited promising antitumor activity and induced tumor cell apoptosis in various cancer cell lines, had previously been reported. Moreover, reports have revealed that the introduction of amino acid to betulinic acid could improve selective cytotoxicity as well as water solubility. Thus, a series of novel TBA amino acid and dipeptide derivatives were designed, synthesized and screened for selective cytotoxic activity against five cancer cell lines (HepG2, HT-29, Hela, BCG-823 and A549) and the not malignant cell line MDCK by standard MTT assay. Most of the tested TBA-amino acid and dipeptide analogues showed stronger anti-proliferative activity against all tested tumor cell lines than TBA. Among them, BA-25 exhibited the greatest cytotoxic activity on tumor cell lines (mean IC50 = 2.31 ± 0.78 μM), that was twofold than the positive drug cisplatin (DDP), while it showed lower cytotoxicity on MDCK cell line than DDP. Further cell apoptosis analyses indicated BA-25-induced apoptosis was associated with loss of mitochondrial membrane potential and increase of intracellular free Ca2+ concentration.

A series of ligustrazine-phenolic acid esters which exhibited promising neuroprotective activities have previously been reported. Nevertheless, we found that these ester compounds (like T-VA) were not stable in plasma by further in vivo studies. To investigate plasma-stable neuroprotective agents, a series of new ligustrazine derivatives were synthesized by conjoining ligustrazine and phenols with ester, ether and amide bonds. Most of the compounds exhibited higher protective effects against CoCl2-induced neurotoxicity in differentiated PC12 cells than ligustrazine. Structure-activity relationships were also briefly discussed. We found that compound 2c (2-((2-methoxy-4-(((3,5,6-trimethylpyrazin-2-yl)methoxy) methyl)phenoxy)methyl)-3,5,6-trimethylpyrazine) displayed the highest protective effect on the PC12 cells damaged by CoCl2 (EC50 = 1.07 μM). Preliminary stability investigation in rat plasma was verified in vitro and better plasma stability was observed with 2c in comparison to T-VA.

The candidate drug T‐VA (C24 H28 N4 O4 ) was synthesized using two kind of neuroprotective ingredients from Chinese traditional medicinal herbs , and displayed promising protective effect on the injured PC12 cells .In previous study ,this beneficial effect was due to the modulation of nuclear transcription factor‐κB/p65 (NF‐κB/p65) and cyclooxygenase‐2 (COX‐2) expressions .T‐VA also exhibited neuroprotective effect in a rat model of ischemic stroke with concomitant improvement of motor functions .Un‐derstanding drug metabolites contributes to discovering and developing the novel drug from the metabolites possessed the pharmacological activities .The structure profile of the metabolites provides an essential perspective for the synthetic refinement and the candidates among an extensive series of potential structures ,resulting in an optimum drug effectiveness and safety .Liquid chromatography with electrospray ionization mass spectrometric detection (LC‐ESI‐MS) has been extensively utilized for the online analy‐sis and structural characterization of the active ingredients and metabolites .Thus ,there is a need to determine the primary metabolites and mass fragmentation pathways of T‐VA in order to understand its potential pharmacological applications .It is a pathway via LC/LTQ‐Orbitrap MS to investigate the mass fragmentation of a candidate drug T‐VA and study its metabolites in rats .As a result ,a method of LC/MSn was estab‐lished for the analysis of T‐VA and its metabolites in rats .The fragmentation pathway of T‐VA was explained using the Analyst V4.0 software .By further analysis of main fragment ions (m/z 317 ,285 ,135) and structural information (C17 H21 O4 N2 ,RDB :8.5 , delta ppm :-3.038 ppm ) , M1 [methyl‐3‐methoxy‐4‐((3 ,5 ,6‐trimethylpyrazin‐2‐yl ) methoxy)benzoate] was discovered as one of the main metabolites .According to the suppositional structure of M1 ,M1‐1 was synthesized via condensation reaction ,which was determined by nuclear magnetic resonance spectrum (1 H‐NMR ,13C‐NMR) and HRMS .By comparing the mass spectrum characters and chromatographic features of M1‐1 and M1 ,it can confirm the exact structure of M1 .Furthermore ,the neuroprotec‐tive effect of M1 in differentiated PC12 cells were evaluated .As a result ,M1 in differ‐ent concentrations could protect PC12 cells injured by CoCl2 (EC50 = 16.01 μmol/L ) , which was close to T‐VA .The result indicated that both T‐VA and its main metabolite have neuroprotective effect ,w hich provides references for further new drug design .In this study ,metabolite of candidate drug T‐VA was obtained by chemical synthesis and verified by MS technique ,which provided a novel idea on the study of drug metabolism . M1 may become a potential neuroprotective agent and further studies are currently underw ay .

Ligustrazine-vanillic acid derivatives had been reported to exhibit promising neuroprotective activities. In our continuous effort to develop new ligustrazine derivatives with neuroprotective effects, we attempted the synthesis of several ligustrazine-vanillic acid amide derivatives and screened their protective effect on the injured PC12 cells damaged by CoCl 2 . The results showed that most of the newly synthesized derivatives exhibited higher activity than ligustrazine, of which, compound VA-06 displayed the highest potency with EC 50 values of 17.39 ± 1.34 μM. Structure-activity relationships were briefly discussed. Graphical abstract New series of ligustrazine-vanillic acid amide derivatives were synthesized and evaluated for their protective effect on the injured PC 12 cells damaged by CoCl 2 . VA-06 was found to be the most active one

目的 建立以γ-氨基丁酸转氨酶(GABA-T)抑制剂类抗癫痫神经系统药物筛选模型,并运用此模型对天麻有效成分及其类似物进行体外活性筛选和构效关系分析.方法 优化酶催化反应温度、反应时间、底物NAD+浓度、底物α-酮戊二酸浓度、底物γ-氨基丁酸(GABA)浓度等因素建立GABA-T酶系活性筛选模型,采用对羟基苯甲醛(HBA)及其11种结构类似物验证模型可靠性.结果 成功构建GABA-T酶系抗癫痫活性筛选模型,HBA及1 1个结构类似物测定结果与文献报道抗癫痫作用相一致,且构效分析发现-OH及苯环对位上的-CHO为必需药效团.结论 建立的模型可应用于GABA-T酶抑制剂的高通量筛选,为GABA-T酶抑制剂类抗癫痫活性成分的筛选及其作用机制研究提供参考.

Compounds in the form of precipitation (CFP) are universally formed during the decocting of Chinese prescriptions, such as Huang-Lian-Jie-Du-Tang (HLJDT). The formation rate of HLJDT CFP even reached 2.63% ± 0.20%. The identification by liquid chromatography mass spectrometry (LC-MSn) proved that the main chemical substances of HLJDT CFP are baicalin and berberine, which is coincident with the theory that the CFP might derive from interaction between acidic and basic compounds. To investigate the formation mechanism of HLJDT CFP, baicalin and berberine were selected to synthesize a simulated precipitation and then the baicalin–berberine complex was obtained. Results indicated that the melting point of the complex interposed between baicalin and berberine, and the UV absorption, was different from the mother material. In addition, 1H-NMR integral and high-resolution mass spectroscopy (HR-MS) can validate that the binding ratio was 1:1. Compared with baicalin, the chemical shifts of H and C on glucuronide had undergone significant changes by 1H-, 13C-NMR, which proved that electron transfer occurred between the carboxylic proton and the lone pair of electrons on the N atom. Both HLJDT CFP and the baicalin–berberine complex showed protective effects against cobalt chloride-induced neurotoxicity in differentiated PC12 cells. It is a novel idea, studying the material foundation of CFP in Chinese prescriptions.

Glycyrrhetinic acid (GA) derivatives had shown not only cytotoxicity but also could trigger apoptosis in various human cancer cell lines. Moreover, cinnamic acid (CA) and its phenolic analogues as potent antitumor agents were employed in the design of anti-tumor drugs. To further improve the anti-tumor activity of GA and CA derivatives, a series of novel compounds were designed and synthesized using GA and CA derivatives fragments.

Objective To establish a model of screening succinate semialdehyde dehydrogenase (SSADH)inhibitors with anti-epilepsy effects,then using this model to find out the effective components in Tall Gastrodia Tuber (Latin:Rhizoma Gastrodiae,pinyin:Tianma)and some analogues and to study their structure-activity relationship.Methods First,SSADH enzyme system fluid was prepared,and the correlation of activity and optical density of SSADH was evaluated using UV spectrophotometry.Then,af-ter the screening method of SSADH activity was established by optimization of reaction temperature,reac-tion time,concentration of NAD +,SSA and SSADH,and pH of buffer on the SSADH activity,p-hydroxybenzaldehyde (HBA),a positive drug recorded in literature,was applied to verify the model. And the structural analogues of HBA were measured and the mechanism was analyzed.Results The screening model was established successfully,proved by HBA test.The UV detective system and its buff-ers of SSADH activity were determined with reaction temperature at 37 ℃ for 30 min,and detect wave at 340 nm.The structure-activity of HBA on GABA-T was as the same as that of vanillic aldehyde,and—OH and —CHO of benzene ring were the essential groups for inhibition SSADH.Conclusion The model established in this paper can be used for high throughput screening SSADH inhibitors and may guide the study of analogues of SSADH inhibitors and their mechanism.