Search Results (2,561)

Background: This study investigated the effects of increasing levels of salinity on leaf phytochemical composition and the antioxidant, antidiabetic, and anti-obesity activities. Method: Three quinoa accessions grown under escalating NaCl treatments had their leaves exposed to various chemical analyses. Polyphenols, tannins, and flavonoids were among the phenolic substances whose concentrations were measured. The phenolic chemicals in the water extract were identified using HPLC-DAD-ESI-MS/MS. In vitro and in silico methods were used to measure anti-radical (DPPH), anti-alpha glucosidase, anti-alpha-amylase, and anti-lipase activities. Results: The results showed that water and ethanol, due to their polarity, were the most effective solvents for extracting phenolic compounds. Additionally, salt application led to a dose-dependent increase in total phenols (TPC), flavonoids (TFC), and tannins (CT) across all accessions. The accession DE-1 exhibited the highest contents with average values of 1453.03–4398.36 mg EGA/100 g DW, 322.7–1090.7 mg CAE/100 g DW, and 77.9–335.96 mg CAE/100 g DW of TPC, TFC, and Tannins, respectively. HPLC-ESI-DAD-MS/MS profiling of phenolic compounds led to identifying 18 constituents, including five major compounds (p-coumaric acid, caffeic acid, vanillic acid, p-coumaroyl hexose, and HHDP-galloyl glucose). Except for p-coumaroyl hexose and HHDP-galloyl glucose, which were extensively biosynthesized/accumulated in the salt-tolerant accession DE-1, the remaining phenolic compounds showed irregular evolution depending on accession and salt concentration. Moreover, ethanol and water extracts were evaluated for their anti-radical and enzyme-inhibitory activities. Conclusion: Salt-stressed DE-1 water extract showed strong antioxidant and enzyme inhibitory activities, indicating potential antidiabetic and anti-obesity effects. These activities were confirmed by in silico analysis. Full article

Background: Neuroinflammation is recognized as a key contributor to Parkinson’s disease (PD), but the relationships between inflammatory signaling, immune-state alterations, and cell-type-specific transcriptional programs remain unclear. Methods: Public transcriptomic datasets, including GSE20141 (discovery cohort) and the substantia nigra subset of GSE114517 (external validation cohort), were analyzed. Genes identified by exploratory differential-expression screening in the discovery cohort were intersected with predefined inflammation- and chemokine-related gene sets to define a candidate space for downstream prioritization. Protein–protein interaction, Gene Ontology, KEGG, and immune-signature analyses were performed, followed by machine learning-based feature prioritization using Elastic Net, support vector machine-recursive feature elimination, and random forest. Prioritized candidates were further evaluated by cross-platform validation, single-nucleus transcriptomic mapping, and a hypothesis-generating in silico perturbation analysis in PD astrocytes. Results: Seventeen genes were retained at the intersection of PD-related differentially expressed genes and inflammation-/chemokine-associated gene sets. These candidates formed a response module enriched in mitochondrial organization, oxidative phosphorylation, and mitophagy pathways. Immune-signature analysis suggested an altered transcriptome-derived immune landscape in PD, with changes in NK cell-related signatures and significant correlations between immune-state scores and the candidate genes. Machine learning-based prioritization yielded five shared candidates, of which only CCL2 and PAK6 showed same-direction support with nominal significance in the external validation cohort. Single-nucleus transcriptomic analysis localized CCL2 predominantly to astrocytes, whereas PAK6 was more strongly associated with neuronal populations, particularly OTX2-positive ventral midbrain neurons. In silico perturbation analysis further predicted that CCL2 suppression in PD astrocytes may be associated with translational- and ribosome-related regulatory programs. Conclusions: CCL2 and PAK6 emerged as prioritized candidate biomarkers associated with PD-related inflammatory and chemokine-linked transcriptional alterations in the substantia nigra. More broadly, this study provides a multi-layered framework for candidate prioritization, cross-platform validation, and cell-type-level contextualization in PD neuroinflammation. Because the study is computational and the perturbation analysis is predictive, orthogonal experimental validation will be required to determine whether CCL2 and PAK6 are biomarkers of disease-associated transcriptional states, functional contributors to PD pathogenesis, or both. Full article

Plant-derived sweet proteins are promising low-calorie natural sweeteners that may reduce dietary sugar intake and prevent non-communicable diseases. Although seven have been identified—thaumatin, miraculin, monellin, mabinlin, brazzein, pentadin, and curculin (neoculin)—only thaumatin is currently approved as a food additive. The development of others requires comprehensive safety assessments, particularly regarding allergenicity. This systematic review aims to investigate and synthesize allergenicity assessment methods (in silico, in vitro, in vivo, and clinical) applied to these seven sweet proteins. The literature searches were conducted following PRISMA guidelines across Scopus, PubMed, and Wiley Online Library databases, up to 30 November 2025, with no time restrictions. The risk of bias in selected studies was evaluated using GRADE. After the selection process, 14 out of 2634 studies met the inclusion criteria. Thaumatin, miraculin, monellin, and brazzein emerged as the most extensively studied proteins. In silico approaches (sequence and structural homology) and in vitro assays (digestibility and cell-based methods) were the most commonly employed methods. In contrast, in vivo studies (animal models) and clinical evaluations (skin prick tests, oral food challenges) were rarely reported. Allergenicity studies on pentadin, mabinlin, and curculin (neoculin) are limited, indicating a research gap that requires further study to support regulatory approval and consumer acceptance. Full article

Background/Objectives: Neuroinflammation is characterized by the sustained activation of neuroglial cells, resulting in the production of cytokines and chemokines. It is associated with neurodegenerative processes. This study aims to assess the potential mitigating effect of a novel coumarin–quinoline hybrid by evaluating oxidative stress, apoptosis, and pyroptosis in an experimentally induced model of neuroinflammation. Methods: The study was conducted on 60 mice, allocated into six groups of ten: Group I served as the control; Group II received the new coumarin–quinoline hybrid; Group III received lipopolysaccharide (LPS); Group IV received LPS followed by the coumarin–quinoline hybrid; Group V received LPS followed by dexamethasone (DEX); and Group VI received LPS followed by the coumarin–quinoline hybrid and DEX. The model was validated by behavioral assessments, while oxidative stress was quantified via nitric oxide (NO), malondialdehyde (MDA) levels, superoxide dismutase (SOD) activity, apoptosis by caspase-3, and pyroptosis by NLRP3. Results: An anti-inflammatory effect of a new coumarin–quinoline hybrid, evidenced by decreased NLRP3 and NF-κB expression, reduced NO and MDA production, elevated SOD activity, and brought about suppression of caspase-3. Additionally, the newly formulated coumarin–quinoline hybrid demonstrated favorable ADMET characteristics, with in silico molecular studies indicating a stable energetic profile and dynamic equilibrium. Conclusions: Findings suggest that the new coumarin–quinoline hybrid holds significant potential as an adjuvant therapeutic option for neuroinflammation. Full article

Background: ADAMTS13 is a protein that cleaves large multimers of von Willebrand factor, thereby limiting platelet aggregation and adhesion and regulating thrombogenesis. Research findings suggest a possible association between low ADAMTS13 levels and an increased risk of cardiovascular events, and its activity may be influenced by polymorphic variants of the ADAMTS13 gene. Methods: The study group included 259 patients diagnosed with coronary artery disease (CAD) and 238 control blood donors. Genotyping of ADAMTS13 polymorphisms (rs2301612, rs2073932, and rs2285489) was performed using TaqMan PCR. Results: ADAMTS13 gene polymorphisms showed no association with CAD risk or patient survival at 5- or 10-year follow-up. However, higher HDL cholesterol levels were observed in carriers of the G alleles (rs2301612 and rs2073932) and the T allele (rs2285489). Additionally, the rs2285489 and rs2301612 polymorphisms were associated with certain proatherogenic lipid indices. In silico analysis indicated that all studied polymorphisms influenced gene expression in certain vascular tissues or blood. Conclusions: ADAMTS13 gene polymorphisms may affect gene expression in specific tissues; however, this effect does not appear sufficient to meaningfully influence CAD onset or patient survival. A significant association between the analyzed polymorphisms and HDL levels or some proatherogenic lipid indices was observed; however, the underlying mechanism requires further investigation. Full article

Background/Objectives: KRAS G12C mutations define a clinically actionable subset of solid tumors, particularly non–small cell lung cancer. Clinical responses to approved covalent inhibitors remain limited by intrinsic and acquired resistance, highlighting the need for structurally distinct inhibitor scaffolds to expand therapeutic options. The objective of this study was to identify novel covalent binders targeting the KRAS G12C switch-II pocket through large-scale in silico screening and experimental validation. Methods: More than 1.9 million small molecules from diverse commercial libraries were screened using covalent docking, followed by multi-stage refinement incorporating molecular dynamics simulations, MM/GBSA free-energy estimation, and cancer-focused QSAR modeling. Results: This integrated workflow yielded 50 prioritized compounds spanning several chemically distinct scaffold classes. These candidates displayed favorable predicted binding energetics, stable ligand-protein interactions over extended simulation timescales, and low structural similarity to clinically approved KRAS G12C inhibitors sotorasib and adagrasib. Benchmarking against these clinical agents, using identical computational parameters, yielded comparable predicted binding energies for several candidate molecules. In cellular NanoBRET target-engagement assays, selected scaffolds, including K788-7251 and AN-989/14669131, exhibited sub-micromolar engagement of KRAS G12C with minimal endothelial cytotoxicity. Conclusions: Collectively, these findings identify structurally distinct, KRAS G12C inhibitor chemotypes and provide tractable starting points for the development of next-generation targeted therapies. Full article

Background: Intervertebral disc degeneration (IVDD) is driven by the interplay between inflammatory signaling, extracellular matrix (ECM) degradation, and impaired cellular adaptation. Although several nutraceutical compounds have been reported to exert protective effects in IVDD-related models, their multitarget mechanisms within integrated molecular networks remain incompletely characterized. Methods: An in silico framework integrating molecular docking with network-based analyses was employed to evaluate resveratrol, quercetin, melatonin, curcumin, and baicalein against a predefined panel of IVDD-associated targets, within an exploratory in silico framework. Binding affinities and interaction profiles were assessed using molecular docking, followed by protein–protein interaction (PPI) network construction, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, and hub gene identification. Results: Docking analyses revealed binding energies ranging from −4.59 to −13.25 kcal/mol, with curcumin and quercetin showing plausible docking poses across a subset of selected targets under the applied protocol. Network analysis showed a highly interconnected structure centered on key inflammatory regulators, including NFKB1, IL6, TNF, IL1B, STAT3, and NLRP3, together with ECM-associated components such as ACAN, COL2A1, SOX9, MMP13, and ADAMTS5. Enrichment analyses further suggested significant associations with inflammatory signaling pathways, cytokine regulation, and ECM organization. Conclusions: These findings are compatible with a distributed, multitarget interaction pattern of nutraceutical compounds within IVDD-associated molecular networks. By integrating molecular docking with network-based analyses, this study offers a system-level framework for interpreting previously reported effects within a disease-specific context. Docking-derived interaction patterns should be interpreted as qualitative and exploratory observations, as docking scores represent model-dependent estimates and do not establish comparable pharmacological effects across heterogeneous targets. The results should be considered hypothesis-generating and require experimental validation. Full article

We performed a protein-docking study for eight DNA aptamers (SEQ1–SEQ8) against chicken Cluster of Differentiation 40 (chCD40), which were experimentally identified via SELEX in our previous study. In silico and molecular docking analyses were performed to predict and obtain the secondary and tertiary structures of the aptamers. Aptamers SEQ3 and SEQ4, which showed the best inhibitory effects, were selected and utilized to produce a DNA-based vaccine adjuvant using rolling circle amplification (RCA). These aptamers had been previously characterized via mass spectroscopy to determine their molecular weight and regions that could potentially interact with chCD40. In the present study, these results were corroborated and expanded. A series of free software methods, including Mfold v.1.0, 3dADN v.2.0, ClusPro v.2.0, Hdock v.1.0, and PLIP v.1.0, were used to determine the aptamers’ secondary and tertiary structures and docking interactions, as well as the specific residues involved in the interactions and their distances. The structures were used to explain and thus understand their effect on the binding, selectivity, and stability of the aptamers. The main objective of the study was to determine whether these aptamers could be used as vaccine adjuvants against viral and bacterial pathogens, specifically chicken avian influenza. The docking results were in good agreement with the experimental and biological results. The procedure employed in this study could be an easy and effective tool for exploring the potential of the new technology of systematic evolution of ligands by exponential enrichment (SELEX) in the preparation of aptamers to control viral and bacterial infections as well as diseases, such as cancer and Alzheimer’s. Full article

Background/Objectives: Rebamipide is a gastroprotective agent with poor aqueous solubility and rapid gastrointestinal clearance, leading to reduced therapeutic efficiency. This study aimed to enhance the solubility, mucoadhesion, and sustained oral delivery of Rebamipide through the development of a deep eutectic mixture (DEM)-based bioadhesive controlled-release granule formulation. Methods: In silico hydrogen-bonding interactions between Rebamipide, malonic acid, and urea were analyzed using CCDC tools. A thermodynamically stable DEM (1:3:1) was prepared and incorporated into bioadhesive granules using chitosan and HPMC. Physicochemical characterization was conducted using FTIR, DSC, TGA, and PXRD. Solubility, in vitro dissolution, ex vivo mucoadhesion (sheep gastric mucosa), and in vivo gastric retention (BaSO₄-loaded granules in rats) were evaluated. Results: The optimized DEM significantly enhanced Rebamipide solubility (10.08 mg/mL vs. 0.045 mg/mL). Solid-state analyses confirmed hydrogen-bond formation and reduced crystallinity. DEM granules exhibited sustained drug release over 24 h (99.7 ± 0.8%) with improved dissolution efficiency compared to the marketed tablet (Mucosta®, 100 mg; T₅₀%: 5.03 h vs. 0.82 h). Kinetic modeling indicated non-Fickian anomalous transport (n = 0.47). The bioadhesive force of DEM granules (0.29 ± 0.02 N) was significantly higher than that of the pure drug and physical mixture. In vivo radiographic studies confirmed prolonged gastric retention. Conclusions: The DEM-based bioadhesive granule system effectively improves solubility, dissolution rate, mucoadhesion, and gastric retention of Rebamipide. This approach represents a promising platform for once-daily gastroretentive oral delivery, pending further pharmacokinetic evaluation. Full article

Background/Objectives: Due to the widespread problem of antimicrobial resistance (AMR), there is an urgent need to identify new antibacterial targets that act through mechanisms distinct from those of existing antibiotics. One of these targets is the essential cell division protein FtsQ, which is a central hub of the Gram-negative divisome, but the druggability of its extensive protein–protein interaction (PPI) interfaces remains poorly defined. Here, we present a comprehensive structure-based in silico characterization of Escherichia coli FtsQ aimed at identifying and prioritizing druggable regions for PPI modulation. Methods: We analyzed E. coli FtsQ in both apo and complexed states (FtsQB, FtsQL, and FtsQBL) using a combination of pocket-mapping tools (FTMap and SiteMap), evolutionary conservation analysis (ConSurf), and structure property assessment (BLAST, ProBiS) to map and evaluate potential binding pockets of FtsQ protein. Results: Eight potential binding sites were predicted across the β and POTRA domains of FtsQ. One previously unreported site within the POTRA domain was prioritized as a candidate site, characterized by favorable druggability scores, strong evolutionary conservation, and a putative role in the FtsQ–FtsW/FtsN/FtsI interaction network. In contrast, two highly conserved sites at the FtsQ–FtsB/FtsL interaction interface were structurally flat, indicating limited suitability for classical small-molecule binding and greater compatibility with alternative modalities such as macrocycles or peptidomimetics. Conclusions: Although FtsQ lacks deep canonical binding pockets, this study proposes several conserved and potentially tractable regions as candidate sites, supporting its potential as a non-classical but promising antibacterial target for disrupting bacterial cytokinesis. Full article

Diabetic kidney disease (DKD) affects approximately 40% of patients with diabetes mellitus and remains a leading cause of end-stage renal disease worldwide. Early diagnosis and identification of therapeutic targets are critical for improving patient outcomes, yet reliable biomarkers are lacking. This study integrated transcriptomic data from the Gene Expression Omnibus (GEO) database (GSE96804, GSE30528, and GSE142025) with machine learning algorithms and Mendelian randomization (MR) to identify diagnostic biomarkers for DKD. Differentially expressed genes (DEGs) were identified and intersected with key modules from weighted gene co-expression network analysis (WGCNA). Four machine learning methods—least absolute shrinkage and selection operator (LASSO), random forest (RF), support vector machine-recursive feature elimination (SVM-RFE), and extreme gradient boosting (XGBoost)—were applied for feature selection. Five hub genes (SPP1, CD44, VCAM1, C3, and TIMP1) were identified at the intersection of these approaches. Two-sample MR analysis using eQTL data from the eQTLGen Consortium and kidney function GWAS from the CKDGen Consortium provided evidence supporting potential causal associations between SPP1, C3, and TIMP1 expression and estimated glomerular filtration rate decline. Immune infiltration analysis via CIBERSORT estimated elevated proportions of M1 macrophages and activated CD4${}^{+}$ memory T cells in DKD samples, with all five hub genes showing correlations with macrophage infiltration. A diagnostic model based on these five genes achieved a cross-validated area under the receiver operating characteristic curve (CV-AUC) of 0.938 in the discovery dataset and AUC values of 0.917 and 0.889 in two independent external validation cohorts. Drug–gene interaction analysis identified 10 candidate compounds targeting the hub genes. These findings provide a computational framework for identifying candidate diagnostic biomarkers and generating hypotheses regarding potential therapeutic targets for DKD; however, all results are derived from in silico analyses and require experimental validation—including qPCR, immunohistochemistry, and prospective clinical cohort studies—before clinical applicability can be established. Full article

Background: Oxidative liver damage, fibrosis, cirrhosis and liver failure are caused by reactive oxygen species and inflammatory responses triggered by bile retention during prolonged cholestasis. Pomegranate and persimmon fruits, which are loaded with bioactive compounds that have anti-inflammatory and antioxidant properties, were evaluated separately for their efficacy in preventing oxidative stress and inflammation in cholestasis. Methods: Pomegranate and persimmon juices were analyzed for their vitamin C, carotenoids and organic acid levels, phenolic profile, and antioxidant activity. Liver protection against oxidative stress and inflammation brought on by cyclosporine-induced cholestasis in rats was verified by biochemical measurements, metabolite identification, and histopathologic examination. To forecast the mechanism of pomegranate and persimmon anti-inflammatory action, an in silico assessment was also carried out. Results: Vitamin C levels in pomegranate and persimmon juices were 99.55 and 51.75 µg/g, respectively. In both pomegranate and persimmon juices, gallic acid was the most prevalent phenolic compound (123.20 and 50.69 µg/g, respectively). Pomegranate and persimmon juices significantly (p < 0.05) reduced the rise in liver values of MDA, NO, TNF-α, IL-6, IL-1β, and TLR4, as well as serum values of total and direct bilirubin caused by cyclosporine. Additionally, the alteration of metabolites, particularly amino acids, demonstrated the inhibitory effect of pomegranate and persimmon juices on liver damage. Gallic acid’s and catechin’s substantial binding affinities with target inflammatory cytokines (TNF-α and TLR4) were further validated by molecular docking. Conclusions: These results showed that pomegranate and persimmon juices mainly modulated inflammation and oxidative stress to provide hepato-protective benefits against cyclosporine-induced cholestatic liver injury. Full article

Background/Objectives: The most abundant flavonoid, quercetin, which is mostly found as glycosides, is widely distributed in plants. Quercetin is rapidly metabolized, having a short half-life in the blood circulation, and forms its conjugates by undergoing ring cleavage of the benzopyranone ring system. Despite its fast clearance in the body, quercetin was demonstrated to have clinically anti-inflammatory, cardioprotective, antidiabetic, and anti-obesity activities. This study aimed to determine whether quercetin itself or its metabolites are responsible for these activities. Methods: We performed molecular docking of 27 metabolites, including quercetin itself, against ten inflammation-related proteins in silico. We then conducted microscale thermophoresis (MST) of selected metabolites towards the NLRP3 inflammasome. Results: Overall, Phase II metabolites yielded better binding energies compared to the metabolites formed by degradation. MST results revealed that isorhamnetin, the 4-O-methylated metabolite of quercetin, gave the best results, with a binding affinity (K_D value) of 16.12 ± 5.16 µM, even better than quercetin itself, which has a binding affinity of 44.84 ± 4.21 µM. Glucuronide metabolites of quercetin (isorhamnetin 3-O-glucuronide, quercetin 7-O-glucuronide, and quercetin 3-O-glucuronide) were found to bind to the inflammasome protein with low binding affinities, whereas small degradation products (hippuric acid and 3,4-dihydroxytoluene) did not bind at all. Conclusions: These results suggest that Phase II metabolites, specifically isorhamnetin, may contribute more significantly to the biological activity of quercetin than the parent compound, however, degradation products appear inactive. Full article

Background: Prostate cancer (PCa) is the leading male urinary malignancy globally. Our previous article demonstrated the anti-PCa activity of Euphorbia humifusa Willd water extract (EHW) and some of its compounds via downregulating AR expression, but the anti-PCa active compounds from Euphorbia humifusa Willd (EH) and their mechanisms of action are yet to be clarified. Thus, the current article studied the in vitro anti-PCa effects of 3,3′-di-O-methylellagic acid (3,3′-di-O-Me-EA) derived from EHW and the related mechanism involved. Methods: 3,3’-di-O-Me-EA was isolated from EHW applying bioassay-guided fractionation. The spectroscopic methods were used to determining the structure of 3,3′-di-O-Me-EA. The drug-likeness and ADMET properties (absorption, distribution, metabolism, excretion, and toxicity) of 3,3′-di-O-Me-EA were analyzed in silico. Molecular docking and real-time surface plasmon resonance (SPR) analysis were performed to measure the interaction of 3,3′-di-O-Me-EA and VDAC1 protein. The viability and apoptosis of 22RV-1 and DU145 PCa cells were determined using MTT and Annexin V-FITC staining assay, respectively. q-PCR and Western blot experiments were used to analyzing the gene and protein expressions of VDAC1. Results: 3,3′-di-O-Me-EA was isolated and purified from EHW with a purity of ≥90.06%, and its structure was identified by HRTOF mass, NMR, and an authentic standard. In silico ADMET analysis indicated its favorable drug-like and pharmacokinetic properties. Molecular docking and SPR results confirmed that 3,3′-di-O-Me-EA could bind with the VDAC1 protein. Moreover, 3,3′-di-O-Me-EA dose- and time-dependently inhibited 22RV-1 and DU145 PCa cell viability, and induced apoptosis in a dose-dependent manner (p < 0.05). RT-qPCR and Western blot results showed that 3,3′-di-O-Me-EA dose-dependently up-regulated VDAC1 gene and protein expression levels in 22RV-1 and DU145 cells (p < 0.05). Meanwhile, in VDAC1-depleted 22RV-1 and DU145 cells, 3,3′-di-O-Me-EA down-regulated VDAC1 gene and protein expression levels, increased cell viability, and inhibited apoptosis compared to 22RV-1 and DU145 cells (p < 0.05). Furthermore, 3,3′-di-O-Me-EA enhanced VDAC1 gene and protein expression levels, inhibited cell viability, and induced apoptosis in VDAC1-overexpressed 22RV-1 and DU145 cells compared with 22RV-1 and DU145 cells (p < 0.05). Overall, EH active compound 3,3′-di-O-Me-EA may inhibit viability and induce apoptosis of 22RV-1 and DU145 PCa cells via up-regulating VDAC1 gene and protein expression levels. Conclusion: The results indicated that the 22RV1 and DU145 PCa cell viability inhibitory effects of 3,3′-di-O-Me-EA isolated from EH may be mediated by induction of apoptosis through up-regulation of VDAC1 gene and protein expression levels. Full article

Background/Objectives: Triple-negative breast cancer (TNBC) is frequently characterized by notably elevated Ki-67 expression, a hallmark of uncontrolled rapid cell-cycle progression. However, the underlying mechanisms remain unclear, leading to limited therapeutic options. Methods: In this study, hub gene was identified through integrated bioinformatic analysis of public datasets (TCGA-BRCA and METABRIC). Subsequent functional validation was performed both in vitro and in vivo using siRNA-mediated knockdown and small-molecule inhibitors. Phenotypic effects—including cell viability, cell cycle distribution, DNA synthesis, and clonogenic survival—were comprehensively assessed using MTT assays, flow cytometry, EdU, and colony formation assays. Protein-level changes were confirmed by Western blotting and immunohistochemistry (IHC). To dissect the transcriptional regulation of the key hub gene TTK, we first predicted potential upstream transcription factors using the JASPAR database; binding specificity was then validated through in silico motif analysis, luciferase reporter assays, and chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR). Results: The mitotic kinase TTK is significantly overexpressed in TNBC compared with non-TNBC breast cancers. Notably, TTK overexpression exhibited a strong positive correlation with elevated Ki-67 indices and reduced overall survival in TNBC patients. Functional validation demonstrated that pharmacological or genetic inhibition of TTK effectively induced G2/M cell-cycle arrest and potently suppressed TNBC proliferation in both in vitro cell cultures and in vivo xenograft models. Mechanistically, TTK overexpression stems from enhanced transcriptional initiation driven by the transcription factor NFYA binding to the CCAAT box in the TTK promoter—an interaction newly identified here. Concurrently, TTK blockade disrupted spindle assembly checkpoint (SAC) signaling via BUB1B/MAD1L1 downregulation, triggering mitotic arrest and catastrophe. Conclusions: Collectively, these findings establish TTK as a key cell-cycle regulator driving TNBC proliferation. More importantly, targeting mitotic control through TTK inhibition represents an efficient strategy to impede the aberrantly fast cell cycle progression in TNBC. Full article