Hypoxia is a key feature of the tumor microenvironment that leads to the failure of many chemotherapies and induces more aggressive and resistant cancer phenotypes. Up to date, there are very few compounds and treatments that can target hypoxia. BE-43547A2 from Streptomyces sp. was one of the most hypoxia-selective compounds against PANC-1, MCF-7, and K562 cell lines. In this study, we investigated the molecular mechanism underlying the hypoxia selectivity of BE-43547A2 in human pancreatic cancer cells. We showed that BE-43547A2 displayed hypoxia-selective cytotoxicity in five pancreatic cancer cells (PANC-1, Capan-2, MIA PaCa-2, AsPC-1, and PaTu8988T) with IC50 values under hypoxia considerably lower than those under normoxia. We demonstrated that BE-43547A2 is directly bound to eEF1A1 protein in PaTu8988T cells under hypoxia. Furthermore, we revealed that hypoxia significantly elevated the expression levels of HIF1α, FoxO1, and eEF1A1 in the five pancreatic cancer cells; eEF1A1 interacted with FoxO1 in the cytoplasm, which was disrupted by BE-43547A2 followed by the nuclear translocation of FoxO1 and ultimate inhibition of JAK/STAT3 signaling pathway under hypoxia. This study reveals that BE-43547A2, targeting eEF1A1, disrupts its interaction with FoxO1 in human pancreatic cancer cells under hypoxia. This compound could serve as a potential hypoxia-selective therapy.

ABSTRACT Background Improving the “cold” tumor immune microenvironment (TIME) of small‐cell lung cancer (SCLC) represents a promising therapeutic approach. The metabolite lactate plays a crucial role in shaping the immune‐cold tumor microenvironment (TME) and facilitating tumor progression. Phosphoglycerate kinase 1 (PGK1) is a key enzyme involved in tumor lactate metabolism. This study demonstrates that ACT001 improves the TIME of SCLC through inhibiting lactate production by targeting PGK1. Methods The cytotoxic effects of ACT001 on SCLC cell lines NCI‐H1688 and NCI‐H446 were evaluated using MTT assay, clone formation, EdU incorporation, wound healing, and invasion assays. To elucidate the mechanism of action of ACT001, proteomic techniques, pull‐down assays, LC–MS/MS, surface plasmon resonance, immunofluorescence, lactate generation, glucose uptake, and western blot assays were conducted. A xenograft model was used to assess the in vivo anti‐tumor activity of ACT001. Results ACT001 inhibited the proliferation, invasion, and metastasis of SCLC both in vitro and in vivo. Additionally, it reduced lactate accumulation and M2 macrophage polarization. Mechanistically, ACT001 released micheliolide, which covalently modified Cys316 of PGK1 under physiological conditions. This suppressed PGK1 activity and restored the distribution of PGK1 in mitochondria and the cytoplasm under hypoxic conditions. Conclusions ACT001 inhibits the malignant progression of SCLC by suppressing lactate production, modulating macrophage polarization, and restraining tumor metastasis through PGK1 targeting.

A self-assembling nanoplatform based on FOB (a heteropolysaccharide from Fomitopsis officinalis) named QU@FOB-FcDBA was developed in this study. A dynamic boronate ester bond, a pH-sensitive covalent linkage formed through reversible dehydration reactions, linked FOB and 1,1'-ferrocenediboronic acid (FcDBA) while encapsulating the hydrophobic drug quercetin (QU). The formulation not only enhanced the water solubility of QU, but also indirectly improved the clinical utilization rate of QU. QU@FOB-FcDBA exhibited unique dual pH/ROS-responsive characteristics, enabling precise recognition and selective response to the acidic microenvironment and elevated reactive oxygen species (ROS) levels in tumor tissues. This ensured the accurate and controlled release of QU and FcDBA, achieving synergistic deep penetration and enhanced therapeutic efficacy through the combined induction of ferroptosis and chemotherapy. In vivo, QU@FOB-FcDBA effectively suppressed tumor progression and metastasis. Mechanistically, it induced cell death via multiple pathways: triggering ferroptosis through ROS accumulation, lipid peroxidation (LPO), and glutathione (GSH) depletion, as well as inducing apoptosis. In vitro, FOB upregulated macrophage surface markers (CD40, CD80, CD86, MHC-II) and nitric oxide (NO) expression. This innate immune activation may directly contribute to antitumor effects and establish a favorable immune microenvironment for immunotherapy. These results position QU@FOB-FcDBA as a promising platform integrating ferroptosis induction and chemotherapy for improved cancer treatment.

BACKGROUND:Temozolomide (TMZ), which is an alkylating agent, is the standard chemotherapeutic drug used for glioma treatment. However, the development of resistance to TMZ limits its efficacy. Thus, identifying novel therapeutic targets is necessary. METHODS:In this study, the levels of midkine (MDK) and c-Myc expression in glioma patient samples downloaded from TCGA were analyzed. Their interactions were also demonstrated through microthermometry and immunocoprecipitation. Furthermore, proteomics technology and Western blot showed that MDK interacted with c-Myc and influenced its ubiquitination, thereby activating a prosurvival signalling pathway and epithelial-mesenchymal transition mechanism, which contributed to TMZ resistance. To target the MDK/c-Myc complex, we screened for a small-molecule inhibitor (ACT001) that specifically disrupts the interaction between MDK and c-Myc. Treatment with ACT001 greatly sensitized TMZ-resistant glioma cells to TMZ, promoting cell death and inhibiting cell proliferation. Moreover, combination therapy with ACT001 and TMZ showed synergistic effects that inhibit tumour growth in glioma xenograft models and glioma in situ models. RESULTS:ACT001 facilitated the degradation of c-Myc by focusing on the MDK/c-Myc complex and controlled the Wnt/β-catenin signalling pathway via MDK, ultimately halting the advancement of glioma. When combined with TMZ, ACT001 showed good therapeutic potential for the treatment of glioma. CONCLUSION:Focusing on the MDK/c-Myc complex could be an effective approach to combat resistance to TMZ in glioma. Therapy with ACT001 may be a novel approach to improve the efficacy of TMZ-based chemotherapy in patients with glioma. Further preclinical and clinical studies are warranted to validate the therapeutic potential of targeting the MDK/c-Myc complex in glioma treatment.

Majusculamide D, isolated from the marine cyanobacterium Moorea producens, is an anticancer lipopentapeptide consisting of fatty acid, tripeptide, and pyrrolyl proline moieties. In this work, by utilizing a convergent synthetic approach, late-stage modification, and bioisostere strategy, 26 majusculamide D analogues were synthesized, and two (1i and 1j) demonstrated IC50 values < 1 nM against PANC-1 cancer cells. The results summarized a preliminary structure-activity relationship mainly at the C23, C4, C34, and C10 sites. A series of in vitro assays, including wound healing, transwell, clone formation, EdU, and western blot, confirmed that majusculamide D inhibited the migration, invasion, and proliferation of pancreatic cancer cells. The optimized fluorinated analogue 1n demonstrated a notable enhancement in stability during the mouse plasma assay (>50% left after 24 h), exhibited tumor-suppressive effects (51.5% at a dosage of 5 mg/kg), and successfully mitigated the severe toxicity (no mouse dead) observed in the group treated with majusculamide D (3 mice dead) in a xenografted mouse model.

BACKGROUND:Colorectal cancer (CRC) is the second most common cause of cancer-related mortality and is characterised by extensive invasive and metastatic potential. Previous studies have shown that vitexicarpin extracted from the fruits of Vitex rotundifolia can impede tumour progression. However, the molecular mechanisms involved in CRC treatment are still not fully established. PURPOSE:Our study aimed to investigate the anticancer activity, targets, and molecular mechanisms of vitexicarpin in CRC hoping to provide novel therapies for patients with CRC. STUDY DESIGN/METHODS:The impact of vitexicarpin on CRC was assessed through various experiments including MTT, clone formation, EDU, cell cycle, and apoptosis assays, as well as a tumour xenograft model. CETSA, label-free quantitative proteomics, and Biacore were used to identify the vitexicarpin targets. WB, Co-IP, Ubiquitination assay, IF, molecular docking, MST, and cell transfection were used to investigate the mechanism of action of vitexicarpin in CRC cells. Furthermore, we analysed the expression patterns and correlation of target proteins in TCGA and GEPIA datasets and clinical samples. Finally, wound healing, Transwell, tail vein injection model, and tissue section staining were used to demonstrate the antimetastatic effect of vitexicarpin on CRC in vitro and in vivo. RESULTS:Our findings demonstrated that vitexicarpin exhibits anticancer activity by directly binding to inosine monophosphate dehydrogenase 2 (IMPDH2) and that it promotes c-Myc ubiquitination by disrupting the interaction between IMPDH2 and c-Myc, leading to epithelial-mesenchymal transition (EMT) inhibition. Vitexicarpin hinders the migration and invasion of CRC cells by reversing EMT both in vitro and in vivo. Additionally, these results were validated by the overexpression and knockdown of IMPDH2 in CRC cells. CONCLUSION:These results demonstrated that vitexicarpin regulates the interaction between IMPDH2 and c-Myc to inhibit CRC proliferation and metastasis both in vitro and in vivo. These discoveries introduce potential molecular targets for CRC treatment and shed light on new mechanisms for c-Myc regulation in tumours.

Comprehensive Summary The total synthesis of majusculamide D ( 1 ) was achieved from commercially available materials. In addition, we synthesized eight analogues including three stereoisomers of majusculamide D that differ in the fatty acid chain. Six analogues including a simplified analogue 29 exhibited significant nanomolar‐level IC 50 values against Panc‐1 cells in MTT assays. A preliminary SAR analysis indicated that the hydroxyl group at C 10 and C 2− C 3 unsaturated double bond of majusculamide D were essential in maintaining the high activity against Panc‐1 cells and the orientation of C 40‐Me and C 42‐Me groups was tolerable.

PAI-1 plays significant roles in cancer occurrence, relapse and multidrug resistance and is highly expressed in tumours. ACT001, which is currently in phase I clinical trials for the treatment of glioblastoma (GBM). However, the detailed molecular mechanism of ACT001 is still unclear. In this study, we investigated the effects of ACT001 on glioma cell proliferation and clarified its mechanism. We discovered that PAI-1 was the direct target of ACT001 by a cellular thermal shift assay. Then, the interaction between ACT001 and PAI-1 was verified by Biacore assays, thermal stability assays and ACT001 probe assays. Furthermore, from the proteomic analysis, we found that ACT001 directly binds PAI-1 to inhibit the PI3K/AKT pathway, which induces the inhibition of glioma cell proliferation, invasion and migration. Moreover, the combination of ACT001 and cisplatin showed a synergistic effect on the inhibition of glioma in vitro and in vivo. In conclusion, our findings demonstrate that PAI-1 is a new target of ACT001, the inhibition of PAI-1 induces glioma inhibition, and ACT001 has a synergistic effect with cisplatin through the inhibition of the PAI-1/PI3K/AKT pathway.

Glioma accounts for a large proportion of cancer, and an effective treatment for this disease is still lacking because of the absence of specific driver molecules. Current challenges in the treatment of glioma are the accurate and timely diagnosis of brain glioma and targeted treatment plans. To investigate the diagnostic biomarkers and prospective role of miRNAs in the tumorigenesis and progression of glioma, we analyzed the expression of miRNAs and key genes in glioma based on The Cancer Genome Atlas database. Of the 701 cases that were downloaded, five were normal and 696 were glioma. Then, 1626 differentially expressed genes were identified, and 173 aberrantly expressed miRNAs were calculated by edgeR. GO and KEGG pathway enrichment analyses were performed using Cytoscape software. A coexpression network was built by weighted correlation network analysis (WGCNA). A cell scratch test and transwell, cell apoptosis and cell cycle assays were performed to validate the function of hsa-let-7b-5p. Based on crosstalk genes in the KEGG, PPI network, and WGCNA analyses, PLK1, CCNA2, cyclin B2 (CCNB2), and AURKA were screened as candidate diagnostic marker genes. The survival analysis revealed that high mRNA expression of PLK1, CCNA2, and AURKA was significantly associated with poor overall survival. Furthermore, hsa-let-7b-5p was identified as a core miRNA in the regulation of candidate genes involved in glioma development. We confirmed that hsa-let-7b-5p could inhibit the migration, invasion, and cell cycle of glioma cells. This study provides four potential biomarkers for the diagnosis of glioma, offers a potential explanation of its pathogenesis, and proposes hsa-let-7b-5p as a therapeutic target.

For the chromatographic analysis of biological samples, sample preparation requires efficient matrix removal and retention of the analytes. The development of online pretreatment technologies provides a fully automated solution for biological sample analysis. Online pretreatment solutions improve both throughput and precision. In this study, we compared two online extraction systems, an online solid-phase extraction (SPE) system and an online turbulent flow chromatography (TFC) extraction system, which are being applied by more analysts at present. The comparison showed that the TFC extraction produced better matrix removal effects, while, peak analysis showed that the online SPE system had obvious advantages in peak shape and efficiency. Thus, we developed an automated online SPE-high performance liquid chromatography (HPLC)-diode array detector (DAD) method for the simultaneous determination of ten β-amino alcohols. The results provide a reference for analysts to choose an appropriate online pretreatment method and provide a solution for biological sample analysis of β-amino alcohol drugs.

This study investigated pharmacokinetics, tissue distribution and excretion of ACT001 in Sprague-Dawley rats. Stability study and metabolism study of ACT001 are conducted. The absolute bioavailability of ACT001 is 50.82%. ACT001 has no accumulation effect and displayed wide tissue distribution. ACT001 can be rapidly distributed to tissues after oral administration and can diffuse through the blood-brain barrier. The total cumulative excretion of ACT001 in feces, urine and bile were found to be 0.05, 3.42 and 0.012%, respectively. UPLC/ESI-QTOF-MS coupled with MetaboLynx XS software was utilized to detect the metabolites of ACT001 in vitro. Five metabolites (M1, M2, M3, M4 and M5) were detected. M2 wasn't discovered in human liver microsome samples and bile samples. M1 and M2 weren't discovered in rat plasma and human plasma. M3, M4 and M5 weren't discovered in bile samples. M5 is an active metabolite named micheliolide (MCL). There is no significant difference in half-life, type of identified metabolites and the amount of each metabolites between using rat plasma and human plasma. Owing to the species differences of hepatomicrosome enzymes, significant differences were shown in half-life, type of identified metabolites and the amount of each metabolites between using rat liver microsome and human liver microsome.

This work mainly demonstrated how the physicochemical properties of a click-based lead compound would be affected by the replacement of its triazole ring with a pyrazole or an imidazole ring. Compound A1, a click-based lead from our previous work, was a selective and moderate inhibitor against VEGFR2. Eight new derivatives of A1 were synthesized, from which a pyrazole derivative B2 was selected as a new lead. B2 maintained the in vitro activity of A1. The solubilities of B2 at pH 2.0 and pH 7.4 were enhanced to 1310 and 1.7 μg/mL, respectively. Its log D value of 3.4 would favor B2 to be modified with hydrophilic fragments to further improve intestinal solubility in our future work.

The bioanalysis and especially the sample preparation of nucleoside drugs in complex media, such as human plasma, has been challenging due to the high polarity and high solubility of these drugs in water. Online solid phase extraction (SPE) offers significant advantages, such as automation and timesaving. Thus, several types of SPE columns have been developed for compounds with different polarities. In this study, SPE was applied to overcome the issue of sample pretreatment of nucleoside drugs in human plasma, with the final aim of establishing a robust analytical platform for drugs with similar structures. A simple, easy-to-use, and efficient method is described for the simultaneous determination of lamivudine, zidovudine, didanosine and emtricitabine in human plasma via online SPE and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Following a simple centrifugation step, a 10μL plasma sample was injected directly onto the HPLC system. The Oasis MCX cartridge was washed, and the analytes were removed by back-flushing directly onto the analytical column. The analytes were quantified using a triple-quadrupole tandem mass spectrometer in multiple-reaction monitoring mode. Similarly, with the development and application of a Bond Elut phenylboronic acid (PBA) SPE cartridge, a fully automated online SPE-HPLC-MS/MS method was established for the simultaneous determination of ribavirin and taribavirin in human plasma. Linear calibration curves were obtained over the range of 0.5-2000ngmL-1, and the limit of quantification ranged from 0.5ngmL-1 to 10ngmL-1, which is sensitive enough for clinical drug monitoring. The intra- and inter-day precisions were in the range of 0.2-8.9%, and the trueness ranged between 88.9% and 113.1%. Excellent recoveries from plasma were achieved with a range between 86.7% and 105.1%. This procedure is easier to perform and requires less sample handling compared to methods previously described in the literature. This high-throughput method involving the direct injection of plasma samples may provide a practical solution for the analysis of multiple nucleoside drugs in clinical research. The method was tested in plasma samples from some patients and showed good performance.

A novel parameter of relative recovery (R re ) was defined and determined by online SPE-HPLC to characterize plasma protein binding (PPB) kinetics of highly plasma binding drugs. The proportional relationship of R re with k off of PPB has been established with a new SPE model. A rapid, easy to use method could potentially be used to categorize PK properties of the drug candidates in the decision process of drug discovery and development.

An integration of sample pretreatment automation using online SPE technique could provide an easy to use, efficient, sensitive and high quality methods for TDM.

Nitric oxide (NO) is an important signaling molecule in cardiovascular system, and the sustained release of NO by endothelial cells plays a vital role in maintaining patency and homeostasis. In contrast, lack of endogenous NO in artificial blood vessel is believed to be the main cause of thrombus formation. In this study, enzyme prodrug therapy (EPT) technique was employed to construct a functional vascular graft by immobilization of galactosidase on the graft surface. The enzyme-functionalized grafts exhibited excellent catalytic property in decomposition of the exogenously administrated NO prodrug. Localized and on-demand release of NO was demonstrated by in vitro release assay and fluorescent probe tracing in an ex vivo model. The immobilized enzyme retained catalytic property even after subcutaneous implantation of the grafts for one month. The functional vascular grafts were implanted into the rat abdominal aorta with a 1-month monitoring period. Results showed effective inhibition of thrombus formation in vivo and enhancement of vascular tissue regeneration and remodeling on the grafts. Thus, we create an enzyme-functionalized vascular graft that can catalyze prodrug to release NO locally and sustainably, indicating that this approach may be useful to develop new cell-free vascular grafts for treatment of vascular diseases.

Background There is no highly effective chemotherapy for malignant gliomas to date. We found that dimethylaminomicheliolide (DMAMCL), a selective inhibitor of acute myeloid leukemia (AML) stem/progenitor cells, inhibited the growth of glioma cells. Methods The distribution of DMAMCL in brain was analyzed by an ultraperformance liquid chromatography-mass spectrometry (UPLC-MS/MS) system. The anti-tumor evaluations of DMAMCL in vitro were performed by MTT, FACS and RT-PCR. In vivo, the mixture of C6 cells and matrigel was injected into caudatum, and the anti-tumor activity of DMAMCL was evaluated by tumor growth and rat survival. The toxicity of DMAMCL was evaluated by body weight, daily food intake, hematological or serum biochemical analyses, and histological appearance of tissues. Results The IC50 values of DMAMCL against the C6 and U-87MG cell lines in vitro were 27.18 ± 1.89 μM and 20.58 ± 1.61 μM, respectively. DAMMCL down-regulated the anti-apoptosis gene Bcl-2 and increased apoptosis in C6 and U-87MG cells in a dose-dependent manner. In a C6 rat tumor model, daily administration of DMAMCL for 21 days reduced the burden of C6 tumors by 60% to 88% compared to controls, and more than doubled the mean lifespan of tumor-bearing rats. Distribution analysis showed that the DMAMCL concentration was higher in the brain than in plasma. Evaluations for toxicity revealed that oral administration of DMAMCL at 200 or 300 mg/kg once a day for 21 days did not result in toxicity. Conclusions These results suggest that DMAMCL is highly promising for the treatment of glioma.

A quantitative strategy towards the detection of sofosbuvir in human plasma was developed with online-SPE-LC-HRMS using t-MS2, t-SIM and F-SIM modes.

Inspired by the biosynthesis of sesquiterpene lactones (SLs), herein we report the asymmetric total synthesis of the germacrane ring (24). The synthetic strategy features a selective aldol reaction between β,γ-unsaturated chiral sulfonylamide 15a and aldehyde 13, as well as the intramolecular α-alkylation of sulfone 21 to construct a 10-membered carbocylic ring. The key intermediate 24 can be used to prepare the natural products costunolide and parthenolide (PTL), which are the key precursors for transformation into other SLs. Furthermore, the described synthetic sequences are amenable to the total synthesis of SL analogues, such as trifluoromethylated analogues 32 and 45. Analogues 32 and 45 maintained high activities against a series of cancer cell lines compared to their parent PTL and costunolide, respectively. In addition, 32 showed enhanced tolerance to acidic media compared with PTL. To our surprise, PTL and 32 showed comparable half-lives in rat plasma and in the presence of human liver microsomes.

An on-line solid phase extraction-high performance liquid chromatography (SPE-HPLC) system was developed for the plasma pharmacokinetic study of highly active anti-cancer compound TEB-415 in mouse. A C18 column (Venusil MP, 100 mm × 4.6 mm, 5 μm) was used as the analytical column and a mixed solution of acetonitrile-5 mM monopotassium phosphate buffer (pH 3.5) at a flow rate of 1.0 mL min−1 as the mobile phase for the isocratic elution. The on-line solid phase extraction was carried out on an MF Ph-1 column (10 mm × 4 mm, 5 μm) with purified water as the washing solvent and 5 mM monopotassium phosphate buffer (pH 3.5) as the elution solvent. The detected wavelength was set at 262 nm. The pharmacokinetic parameters were calculated by WinNonlin 5.2 software. The linear range of TEB-415 was 100–20000 μg L−1 with a limit of qualification of 20 μg L−1. The extraction recovery was 91.99%–99.14% and the RSD of intra- and inter-day precision was less than 3.5%. The accuracy of short-term stability, freeze-thaw stability and long-term stability were 91.5%–101.5%. After oral medication, the mean peak time (Tmax) of TEB-415 in mice was 5.29 h and the mean maximum concentration (Cmax) was 3403 μg L−1. The area under the curve (AUC) of TEB-415 was 24600 μg L−1 h−1. The mean half-life of drug in mice was 3.84 h, and the mean retention time (MRT) was 6.56 h. These parameters above suggest that TEB-415 has an appropriate rate of absorption and elimination for the mice. Meanwhile, it had preferable bioavailability.