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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.

Based on the surface plasmon resonance (SPR) technique, the proposed biosensor is investigated as an SPR-based sensing platform for detecting breast cancer cells, specifically MCF-7 and MDA-MB-231 cells. Developed biosensor features an octagonal cylinder-shaped resonator design composed of two novel materials: an octagon-shaped structure made of gold (Au) and a cylinder-shaped structure made of silver (Ag). Graphene is also integrated to achieve high sensitivity in the detection for the two breast cancer cell lines based on the refractive index values, within the wavelength range of 1650-1700 nm, yielding sensitivity rates of 714.28 nm/RIU (MCF-7) and 785.71 nm/RIU (MDA-MB-231). The proposed Graphene Octagonal Cylinder-Shaped Surface Plasmon Resonance (GOCSPR) biosensor consists of two layers, with a ceramic substrate made of aluminum nitride (AlN), and exhibits good quality factors of 560 for MCF-7 and 557 for MDA-MB-231. The analysis of layer height and cylinder radius, along with optimization using a Linear Regression machine learning algorithm and the corresponding R2 values, is also presented in the manuscript. The designed graphene-based structure can be used for detecting breast cancer cell with high efficiency for medical applications.

Targeting autophagy initiation represents a promising strategy to disrupt the metabolic resilience of cancer cells. In this study, we identified ATI-1 as a novel small-molecule inhibitor that selectively blocks the early stages of autophagosome formation. Importantly, we discovered that ATI-1-mediated de novo inhibition of autophagy initiation leads to a synergistic surge in cell death under nutrient-deprived conditions, revealing a critical, context-specific vulnerability in autophagy-dependent malignancies. Mechanistically, ATI-1 appears to target valosin-containing protein (VCP/p97) and disrupt its interaction with the UFM1-specific E3 ligase UFL1. This disruption may promote the polyubiquitination and subsequent degradation of Beclin1, thereby contributing to the inhibition of autophagy initiation. Furthermore, ATI-1 demonstrates potent antitumor efficacy in xenograft models with minimal overt toxicity. This work collectively suggests that the VCP-UFL1-Beclin1 axis may represent a potentially targetable node in autophagy regulation, and identifies ATI-1 as a potential small-molecule modulator of this pathway, thereby providing a promising therapeutic lead for cancer treatment.

Dual inhibition of the epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor-2 (VEGFR-2) represents an effective strategy for achieving synergistic antitumor activity by simultaneously suppressing tumor proliferation and angiogenesis. In this study, a series of quinoxaline-based derivatives (7-15) was rationally designed, synthesized, and biologically evaluated as dual EGFR/VEGFR-2 inhibitors. In vitro cytotoxicity screening against breast (MCF-7), liver (HepG2), and colon (HCT116) cancer cell lines identified compounds 9, 11, 12, and 13 as the most potent antiproliferative agents, exhibiting activities comparable to or exceeding those of the reference drugs Sorafenib and Erlotinib. Among them, compound 12 demonstrated the highest potency, as evidenced by cellular fold inhibition values of 0.288 and 0.227 against EGFR and VEGFR-2, respectively. Enzymatic assays further confirmed its strong inhibitory activity, with IC₅₀ values of 0.06 μM (EGFR) and 0.204 μM (VEGFR-2), comparable to Erlotinib (0.052 μM) and Sorafenib (0.131 μM). Mechanistic investigations revealed that compound 12 exhibited interesting selectivity, with 2.4-fold lower cytotoxicity toward normal MCF10A cells compared to doxorubicin. It induced a pronounced G2/M phase arrest in MCF-7 cells and significantly promoted apoptosis, resulting in a 12-fold increase in apoptotic cell population. Gene expression analysis indicated activation of both intrinsic and extrinsic apoptotic pathways, demonstrated by a marked increase in the Bax/Bcl-2 ratio (∼20-fold) and upregulation of caspase-9 (7.9-fold) and caspase-8 (2.9-fold). Molecular docking studies supported the experimental findings, revealing a strong binding affinity of compound 12 within the active sites of EGFR and VEGFR-2. Molecular dynamics simulations further confirmed the stability of these interactions over time. Additionally, in silico ADME profiling demonstrated good drug-likeness, fulfilling Lipinski, Veber, Egan, and Muegge criteria. Collectively, these findings highlight compound 12 as a promising dual EGFR/VEGFR-2 inhibitor with significant potential for further development as a multitarget anticancer candidate.

The transforming growth factor-β (TGF-β) signaling pathway is crucial in promoting tumor growth, enabling tumors to evade immune responses, and contributing to resistance against therapies. As a result, it is a significant target for cancer treatment. However, its full potential remains untapped because selectively inhibiting it without affecting normal cells is challenging. This study reports the design, synthesis, and comprehensive evaluation of novel benzimidazolium-chalcone hybrid salts (3a-3e) that strategically combine two privileged scaffolds with complementary anticancer mechanisms. Following complete structural characterization by Elemental Analysis, FT-IR, and NMR spectroscopy, an integrated experimental and computational workflow supports a three-strategy hypothesis rather than a single lead compound. Compound 3e emerged as the most selective derivative, showing moderate anti-proliferative activity in U87 cells while maintaining reduced toxicity toward non-cancerous cells. Although direct pathway-level validation was beyond the scope of the present study, the slight reduction observed in extracellular TGF-β1, together with anti-migratory and anti-migratory and anti-clonogenic effects, along with in silico interaction patterns, supports 3e as a promising lead scaffold for further mechanistic investigation. In vitro studies using the glioblastoma cell line, U87, displayed promising anticancer activity of benzimidazolium-chalcone hybrid salts. Compounds 3a, 3b, and 3c demonstrated limited selectivity, with IC₅₀ ratios between cancer and normal cells ranging from 1.5- to 1.7-fold. Compound 3d showed a 1.6-fold and 2.0-fold selectivity advantage over BEAS-2B and HUVEC cells, respectively. In contrast, compound 3e demonstrated the most favorable selectivity profile, with an IC₅₀ of 41.09 μM in U87 cells and IC₅₀ values of ≥96.60 μM in normal cell lines. Molecular docking predicted binding affinities ranging from -9.91 to -11.67 kcal/mol. However, no significant correlation was observed between docking scores and biological activity (R2 = 0.068, p = 0.671). Molecular dynamics simulations (3 × 100 ns) confirmed stable ligand binding for all compounds (protein-ligand minimum distance: ∼0.20 nm), with per-residue energy decomposition revealing that compound 3a binds mainly through extensive hydrophobic contacts (92% van der Waals), while compound 3e forms unique polar interactions with His283 and Tyr282. Principal component analysis revealed distinct conformational profiles (variance: 3a = 0.715, 3d = 1.364, 3e = 0.531), suggesting a possible connection between conformational restriction and cellular safety. ADMET profiling confirmed drug-like properties for compound 3e with no PAINS alerts or CYP3A4 inhibition. These findings support a preliminary hypothesis that links physicochemical properties and interaction quality, rather than static binding affinity, to therapeutic selectivity in TGF-β1 modulation design.

Herein, we report the discovery of APO-50815 (14), a potent and selective thietane-3-ol WEE1 inhibitor. When tested against TP53-mutated colorectal cancer (CRC) patient-derived organoids (PDOs) grown from peritoneal and liver metastases, 14 exhibited outstanding anticancer efficacy, surpassing previously reported branched alkane counterpart 3, as well as clinical candidates AZD1775 (1) and ZN-c3 (2). Against primary CRC organoids with diverse TP53, BRAF and KRAS mutation profiles compared with patient-matched normal colon organoids, 14 exhibited selectively potent activity, yielding exceptionally high TI values (129-238) that highlight a substantial therapeutic window for potential cancer treatment. Against primary CRC PDOs (TP53-WT, BRAF-V600E, KRAS-WT), 14 profoundly elevated DNA damage and replication stress compared to 1, while amplifying cellular apoptosis, confirming a broadly similar but superior mode of action. Owing to its highly selective and exemplary anticancer efficacy, 14 represents a valuable tool compound for drug testing investigations against primary and metastatic CRCs, especially in the context of PDOs.

Triple-negative breast cancer (TNBC) is an aggressive subtype lacking the ER, PR, and HER2 receptors, with limited treatment options and poor prognosis. Ferroptosis, an iron-dependent form of regulated cell death driven by lipid peroxidation, has emerged as a promising therapeutic strategy for TNBC. Glutathione peroxidase 4 (GPX4) is a key ferroptosis suppressor, and its inhibition sensitizes TNBC cells to oxidative damage. To discover and characterize DA-5, a novel 3,5-disubstituted azaindole derivative inspired by compounds from the traditional Chinese medicine Shuganning injection (SGNI), as a potent GPX4 inhibitor to induce ferroptosis in TNBC cells. Molecular docking and structural optimization were used to design DA-5. Its binding affinity (Kd) and enzymatic inhibitory activity (IC50) against GPX4 were evaluated. In vitro assays assessed DA-5's ability to induce ferroptosis in TNBC cells through lipid peroxidation while sparing normal mammary epithelial cells. In vivo studies evaluated the efficacy and safety of DA-5 in TNBC xenograft models via oral administration. Pharmacokinetic profiles were also analyzed. DA-5 demonstrated high-affinity binding to GPX4 (Kd = 10.04 μM) and effectively suppressed its enzymatic activity (IC50 = 10.90 μM). In TNBC cells, DA-5 promoted lipid peroxidation and induced ferroptosis, which was rescued by ferroptosis inhibitors and by iron chelators. Oral administration of DA-5 significantly inhibited TNBC xenograft growth in vivo without systemic toxicity, supported by favorable pharmacokinetic and safety profiles. These findings identify DA-5 as a novel azaindole-based GPX4 inhibitor capable of inducing ferroptosis through GPX4-targeted lipid peroxidation. This breakthrough offers a promising targeted therapy for TNBC.

PI3Kα plays a key role in a variety of cellular processes, and its gene mutations are closely related to the occurrence and development of many types of cancer. Although two orthosteric PI3Kα inhibitors including Alpelisib and Inavolisib have been launched onto market, they often cause toxic and side effects due to insufficient selectivity for PIK3CA mutant protein. The development of allosteric PI3Kα inhibitors provides new ideas for overcoming these problems. Our research focuses on optimizing a novel series of allosteric PI3Kα inhibitors derived from STX-478. By integrating scaffold hopping and comprehensive structural modification strategies, we obtained the lead compound allosteric PI3Kα inhibitor 11f with a benzothiophene scaffold. The inhibitor demonstrates high selectivity for PIK3CA mutant protein, low hERG inhibition, minimal CYP inhibition, excellent in vivo efficacy, good safety and no impact on insulin balance. Collectively, these findings confirm that compound 11f is a highly promising drug candidate for the targeted therapy of PIK3CA H1047R mutant HR+/HER2-breast cancer.

Glioblastoma multiforme (GBM) counts as one of the highly deadly primary intracranial malignancies, posing a significant challenge to clinical management. Focal Adhesion Kinase (FAK) has been identified as a pivotal molecular target implicated in GBM pathogenesis, modulating key processes such as tumor cell proliferation, invasion, and therapeutic resistance. Despite the considerable number of FAK inhibitors advancing to clinical evaluation, their efficacy against GBM remains inadequately documented. In this study, a cyclization strategy was served for discovering novel FAK inhibitors, which was employed TAE-226 as the molecular scaffold. Among the synthesized derivatives, compound 16c distinguished itself as a highly potent inhibitor, showing an IC50 value of 5.8 nM against FAK and robust antiproliferative activities in U87-MG (IC50 = 6.6 nM) and U118-MG (IC50 = 4.3 nM) GBM cell lines. Additionally, 16c exhibited favorable blood-brain barrier penetration, markedly promoted apoptotic cell death, and induced G2/M cell cycle arrest in U87-MG cells. Furthermore, compound 16c exhibited significant inhibitory activity against 25 kinases, which indicated that it could be a promising multi-targeted kinase inhibitor. Importantly, the oral bioavailability of 16c reached 18.7% at a dose of 10 mg/kg, and 16c displayed pronounced antitumor efficacy in the absence of detectable systemic toxicity in a U87-MG xenograft model. These results collectively highlight the promise of FAK inhibition as a therapeutic strategy for GBM.

Accurate subtyping of lung cancer is essential for improving patient prognosis and enabling personalized treatment. However, current clinical techniques are often time-consuming and heavily dependent on the operator's subjective judgment and experience, which limits the accuracy and timeliness of intraoperative subtype diagnosis and margin assessment. In this study, we developed an intelligent diagnostic model by integrating Fourier transform infrared (FTIR) spectroscopy with a Random Forest (RF) classifier. A total of 210 tumor and adjacent tissue samples from 105 patients, including adenocarcinoma, squamous cell carcinoma, and benign lung tumors were analyzed. The constructed RF model achieved an accuracy of 97.95% with an Area Under the Curve (AUC) of 0.99 in binary classification (lung cancer vs. adjacent tissues), and an accuracy of 94.91% in multiclass classification of lung cancer subtypes, significantly outperforming conventional algorithms such as Support Vector Machine, Naive Bayes, and Logistic Regression. In addition, spectral analysis methods, including peak area comparison, peak fitting, and second derivative analysis, revealed distinct differences in nucleic acids, proteins, and lipids, highlighting the characteristic bands responsible for subtype discrimination and providing spectroscopic insights into the pathological features of different lung cancer subtypes. Collectively, our findings demonstrate that the diagnostic model is a powerful approach for distinguishing lung cancer tissues from normal tissues and for subtype classification, offering a promising tool for lung cancer diagnosis.