Lipid nanoparticles (LNPs) are extensively utilized in nucleic acid delivery for therapeutic applications because of their biocompatibility and protection of the nucleic acid cargo from degradation during in vivo transport. The nanoscale DNA-based fluoropyrimidine polymer CF10 shows strong efficacy advantages relative to conventional fluoropyrimidine drugs such as 5-fluorouracil (5-FU). In principle, LNP-mediated delivery of CF10 could further enhance its efficacy advantage relative to 5-FU by increasing plasma stability and promoting cell uptake. However, the anticancer activity of CF10 relies on the release of active nucleotides, and it is not clear that LNP-mediated delivery of CF10 preserves dual targeting of thymidylate synthase (TS) and DNA topoisomerase 1 (Top1). This study proposes the incorporation of CF10 into LNPs using chaotic mixing of lipid compositions in a microfluidic chip. Biophysical characterization revealed homogeneous LNP formation with size 80-200 nm in diameter and a zeta-potential of -15 mV, dependent on CF10 concentration. LNPs were stable (tested in PBS) over 4 weeks. In vitro studies showed that LNP formulation increased CF10 uptake specifically into cancer cells, while an immortalized nonmalignant intestinal cell line did not show increased uptake of CF10:LNPs. CF10:LNPs initially colocalized with endosomes, followed by primarily lysosome colocalization at 48 h. CF10:LNPs displayed increased cytotoxicity to cancer cells relative to free CF10, proportional to increased cell uptake. Potent inhibition of TS was achieved consistently with nuclease-mediated release of FdUMP from CF10 in lysosomes. CF10:LNPs also efficiently induced Top1 cleavage complex formation, consistent with perturbation of cellular dNTP pools similar to free CF10. These findings indicate that CF10:LNPs display enhanced anticancer activity relative to free CF10 and preserve CF10's unique TS/Top1 dual targeting mechanism.

Indane-1,3-dione is a reactive cyclic β-diketone that could be employed for preparing various molecular systems of potential biological applications. Among these, 4- azafluorenones (also known as indeno[1,2-b]pyridines) represent one of the most promising classes of carbocyclic systems. Indeno-fused pyridines possess a wide range of medicinal properties, including anti-proliferative activity and DNA topoisomerase Iα/Iiα inhibitory activity. In this review, we presented all reports from 2000 to 2024 that cover the synthesis of indeno[1,2-b]pyridines and diindeno[1,2-b:2',1'-e]pyridines starting from indane-1,3- dione. The review is classified according to the type of reaction conditions that were applied. Additionally, the reports that are related to the new trends in preparing indenopyridines are indexed in separate sections, including the use of ionic liquids, heterogeneous catalysts, and microwave- and ultrasonic-assisted synthetic routes. Some complex synthetic routes are explained by plausible mechanisms.

Bioorganic synthesis and structure-activity relationships of Origanum vulgare-functionalized zinc oxide nanoparticles: Phytochemical profiling and antimicrobial mechanisms.

Adenoid cystic carcinoma (ACC) is a slow-growing malignant tumour that primarily originates from the major and minor salivary glands. The relationship between the NDC80 kinetochore complex component (NUF2) and ACC remains to be elucidated. The present study obtained gene expression information from the Gene Expression Omnibus database (GSE88804 and GSE153002). Differentially expressed genes were identified by using the ‘limma’ package in R. A protein-protein interaction network was constructed with the Search Tool for Retrieval of Interacting Genes/Proteins database and key genes were extracted using Cytoscape software. Analysis of differential expression levels of hub genes in tumour and normal tissue was performed using Tumour Immune Estimation Resource (TIMER) and GSE36820 profiles. Gene Ontology enrichment was subsequently analysed based on differences in NUF2 expression in tumour tissues. In addition, single sample Gene Set Enrichment Analysis (ssGSEA) was used for the quantitative analysis of immune cell infiltration in ACC. Western blotting and immunohistochemistry were used to assess NUF2 expression levels in tumour and adjacent non-tumour tissues. Small interfering RNA (siRNA) was used to decrease NUF2 expression in ACC cell lines. The biological functions of NUF2 were analysed using Cell Counting Kit-8 and wound healing assays. A total of 248 differential genes were identified by differential expression analyses, with 113 genes upregulated and 135 downregulated. A total of 7 hub genes, namely, CDK1, budding uninhibited by benzimidazoles 1 mitotic checkpoint serine/threonine kinase B, DNA topoisomerase II α, cyclin B2, NUF2, budding uninhibited by benzimidazoles 1 and centromere protein F, were obtained using the ‘cytoHubba’ plugin. The TIMER, standardized and GSE36820 databases revealed that the expression levels of NUF2 were higher in ACC tissues compared with normal tissue samples. Western blotting and immunohistochemical staining of ACC tissues provided evidence of NUF2 upregulation in ACC tissue compared with normal tissue. NUF2-related genes were enriched in ‘ameboidal-type cell migration’, ‘collagen-containing extracellular matrix’ and ‘actin binding’. ssGSEA analysis revealed that the expression level of NUF2 was notably associated with activated CD4+ T cells, memory B cell and plasmacytoid dendritic cell. ACC cells transfected with NUF2 siRNA exhibited decreased proliferation and migration compared with the control. In conclusion, NUF2 is upregulated in ACC and is associated with immune cell infiltration. Functional studies demonstrated that NUF2 promotes ACC cell proliferation and migration, suggesting its potential as a therapeutic target for ACC.

Role of Green-Synthesized Silver and Copper Oxide Nanoparticles in Modulating DNA Topoisomerase III Activity and DNA Methyltransferase I Protein Expression Through In Vitro and In Silico Approaches

BackgroundGepotidacin is a novel, bactericidal, first-in-class triazaacenaphthylene antibacterial that selectively inhibits bacterial replication through a distinct binding site, unique mechanism of action and, for most target pathogens, well-balanced inhibition of two Type II topoisomerase enzymes (DNA gyrase and topoisomerase IV). Gepotidacin was recently approved by the FDA for the treatment of uncomplicated urinary tract infections (uUTI). This study evaluated the effects of several antimicrobial susceptibility testing (AST) variables on standardized (i.e., CLSI, EUCAST) testing methods for key uUTI uropathogens.Variables shown to significantly impact gepotidacin BMD MICs and/or DD zone for EnterobacteralesMethodsThe in vitro activity of gepotidacin and levofloxacin was evaluated using 4 CLSI AST methods: broth microdilution (BMD), broth macrodilution (MD), agar dilution (AD) and disk diffusion (DD). 85 clinical isolates were included for MIC testing and 90 isolates for DD, including E. faecalis (EF), S. saprophyticus (SS), and several Enterobacterales species known to be implicated in uUTI. Variables (BMD and DD only) including but not limited to inoculum concentration, temperature, atmospheric conditions, pH, and urine were tested.ResultsGepotidacin mean reference BMD MICs varied no more than 1 dilution over multiple days, for all species tested. MICs were comparable between the 3 MIC testing methods with MICs for 84 of 85 isolates within ± 1 dilution. Reference gepotidacin mean disk results varied by approximately 1-2 mm over multiple testing days. Mueller Hinton agar data was similar for DD with the exception of SS and Remel in which the majority of results were > 3mm larger. Variables shown to significantly impact gepotidacin BMD MICs and/or DD zone for Enterobacterales are shown in the table below.Results were similar for SS and EF with the exception of EF and SS DD zones being more impacted by inoculum variation and no impact of temperature on EF and SS MICs and DD zones.ConclusionWhen performing susceptibility testing with gepotidacin it is important to control the following variables, which were shown in this study to have the most impact on results: high inoculum concentration, high temperature (for Enterobacterales only), pH, incubation in 5% CO2 (for DD only) and testing of BMD MICs in urine.DisclosuresNicole E. Scangarella-Oman, MS, GSK: Employee|GSK: Stocks/Bonds (Public Company) Josh West, BS, GSK: Employee|GSK: Stocks/Bonds (Public Company)

Background/Objectives: The escalating threat of drug-resistant Neisseria gonorrhoeae underscores the urgent need for novel therapeutic agents. Zoliflodacin, a first-in-class spiropyrimidinetrione antibiotic that targets bacterial DNA gyrase and topoisomerase IV, represents a promising candidate for gonorrhea treatment. Methods: From 2020 to 2023, a total of 876 urogenital N. gonorrhoeae isolates were collected from 35 hospitals across Shanghai, China. In vitro susceptibilities to zoliflodacin and six conventional antibiotics (penicillin, tetracycline, ciprofloxacin, azithromycin, ceftriaxone, and spectinomycin) were determined using the agar dilution method. Whole-genome sequencing was conducted to identify sequence types (STs) and amino-acid substitutions in GyrA, GyrB, ParC, ParE, and MtrR. Results: Zoliflodacin exhibited potent in vitro activity, with minimum inhibitory concentrations (MICs) ranging from ≤0.004 to 0.25 mg/L (MIC50 = 0.06 mg/L; MIC90 = 0.125 mg/L), all below the breakpoint (0.5 mg/L). Notably, zoliflodacin maintained high activity against isolates resistant to ceftriaxone, azithromycin, ciprofloxacin, penicillin, and tetracycline. Although all isolates were susceptible to zoliflodacin, elevated MIC values were observed in ST7363 and ST8123 compared with other clones. Genomic analysis identified no substitutions associated with increased zoliflodacin MICs, and most GyrB sequences, the key gene associated with zoliflodacin resistance, remained intact. Conclusions: These findings demonstrate that zoliflodacin possesses robust activity against circulating multidrug-resistant N. gonorrhoeae lineages in Shanghai and support its potential clinical use for the treatment of gonorrhea. Continued genomic and phenotypic surveillance is warranted to preserve the long-term efficacy of this novel agent.

Synthesis and biological evaluation of phenanthridine derivatives as dual-target inhibitors of DNA topoisomerase IB (TOP1) and tyrosyl-DNA phosphodiesterase 1 (TDP1), and potential antitumor agents.

Preparation and in vitro functional validation of a novel antibody-siRNA-Exatecan conjugate (AREC).

2-Aminobenzothiazole: A Privileged Scaffold for Tyrosine Kinase-Targeted Anticancer Agents.

DNA double-strand breaks induced by DNA topoisomerase IIα and reactive oxygen species lead to the integration of foreign DNA into the host genome.

We aimed to design and synthesize novel pyrimidine-2-thione derivatives (1-13) as Topoisomerase I/II (Topo I/II) inhibitors with DNA intercalation potential for cancer treatment. Inhibitory concentration 50 (IC50) against mammary gland breast cancer, hepatocellular carcinoma, and colorectal carcinoma was determined for all compounds. The frontier candidates (2, 6, 9, 10, and 11) were evaluated for their DNA-binding ability, Topo I, and Topo II inhibiting potential. Moreover, cell cycle and apoptosis analysis were carried out. Compound 2 displayed the best DNA-binding affinity with an IC50 value of 37.24 µM in comparison to doxorubicin (Dox). Both compounds 2 and 9 showed superior nanomolar Topo I inhibitory potential, compared to Dox. Similarly, compounds 2 and 9 achieved better Topo II inhibition, exceeding that of Dox. It was revealed that compound 9 halted the cell cycle at both the P1 and G2 phases. In addition, compound 9 was able to boost the apoptosis at both the early and late apoptotic phases. Consequently, the compounds afforded can be regarded as prominent lead anticancer compounds for further optimization and investigation.

Stepwise DNA damage and repair mechanisms at replication forks in response to topoisomerase I inhibition.

Eukaryotic topoisomerase I (TOP1) plays a crucial role in maintaining DNA structure by relieving torsional stress during essential cellular processes such as replication, transcription, and repair. In humans, TOP1 is a well-established target for anticancer therapies due to its vital functions. Similarly, plant TOP1 is essential for growth, chromatin organization, and stress responses. Arabidopsis thaliana has two Type IB TOP1 enzymes, with TOP1α being highly expressed in rapidly dividing cells and critical for development. While human TOP1 inhibitors, including topoisomerase poisons, have been found to inhibit plant TOP1, there is limited understanding of its structural details in plants. In silico analysis shows significant structural and functional similarities between plant and human topoisomerases, despite differences in their sequences. Functionally, the human TOP1 inhibitor irinotecan significantly reduces root elongation, lowers the mitotic index in root meristems, and suppresses callus proliferation in Arabidopsis seedlings. These findings indicate that rapidly dividing plant cells, especially those overexpressing TOP1α, are susceptible to these topoisomerase poisons. This study provides new insights into the interactions between plant topoisomerases and drugs and suggests A. thaliana as a cost-effective and genetically manageable model for preliminary screening of potential topoisomerase-targeting therapeutics. It also highlights the evolutionary conservation of drug-target interactions, supporting the development of plant-based platforms for discovering new anticancer compounds.

Gene amplification is thought to be common in bacterial populations, providing a rapid and reversible mode of adaptation to diverse stresses, including the acquisition of antibiotic resistance. We previously showed that the opportunistic pathogen Staphylococcus aureus evolves resistance to the dual-targeting fluoroquinolone delafloxacin (DLX) that inhibits both the DNA gyrase and DNA topoisomerase IV via amplification of an efflux pump encoding gene sdrM. However, the pathways that control gene amplification, and consequently adaptive trajectories, remain understudied, especially in gram-positive bacteria like S. aureus. Here, we show that specific DNA repair and chromosomal separation proteins alter the frequency of gene amplification and selection of amplified regions in S. aureus. Through a screen of 40 mutants deficient in various DNA processes, we determined that while sdrM amplification was still the almost universal path to DLX resistance, other mutations that increased sdrM expression reduced the selection frequency of sdrM amplification, demonstrating the critical role of sdrM in DLX resistance. We found that similar to other bacteria, both sdrM amplification and loss of amplified gene copies required a functional RecA recombinase, but multiple other mutants in pathways required for amplification in other species still exhibited frequent sdrM amplification, suggesting that S. aureus may have alternate routes of gene amplification. Finally, loss of function mutants of the tyrosine recombinase XerC, that is known to play a role in chromosomal separation, were deficient for sdrM amplification, indicating that XerC is a novel modulator of gene amplification, or the maintenance or selection of amplified gene copies. Thus, our work sheds light on genetic factors that alter gene amplification-mediated evolutionary trajectories to antibiotic resistance in S. aureus and can potentially unlock mechanisms by which such evolution of resistance can be inhibited.

Pyrano[3,2-c]chromene derivatives, as an important category of heterocyclic compounds, have received much attention due to their various biological activities. Different synthetic methods using various catalysts have been reported for the synthesis of these compounds. Here, a green, magnetically separable catalyst functionalized with nitrogen-containing groups was used for the synthesis of a series of pyrano[3,2-c]chromene derivatives. The target compounds were synthesized by employing the catalytic activity of the previously synthesized composite under environmentally friendly and mild conditions. The reaction proceeded efficiently in a water/ethanol mixture within a short time and resulted in high product yields. A multicomponent reaction was used to synthesize pyrano[3,2-c]chromene derivatives from malononitrile, 4-hydroxycoumarin and various aldehydes. The purity of the final compounds was checked by melting point determination and comparison with literature values. Since pyrano[3,2-c]chromene derivatives are proposed as potential inhibitors of DNA topoisomerases, especially TOPII, in the second part of this study, a computational study was performed targeting the DNA-TOPII complex. The results revealed that some of the synthesized compounds are known to inhibit topoisomerases, supporting their role as promising anticancer agents.

Ubiquitin E3 ligases play crucial roles in the DNA damage response (DDR) by modulating the turnover, localization, activation, and interactions of DDR and DNA replication proteins. We performed a CRISPR-Cas9 knockout screen focused on ubiquitin E3 ligases and related proteins with the DNA topoisomerase I inhibitor camptothecin. This led us to establish that MAEA, a core subunit of the CTLH E3 ligase complex, is a critical regulator of homologous recombination and the replication stress response. In tandem, we identifiedeight patients with variants in MAEA who present with a neurodevelopmental disorder that we term DIADEM (Developmental delay and Intellectual disability Associated with DEfects in MAEA). Analysis of patient-derived cell lines and mutation modeling reveal an underlying defect in HR-dependent DNA repair and replication fork restart and protection as a likely cause of disease. Mechanistically, we find that MAEA dysfunction hinders DNA repair by reducing the efficiency of RAD51 loading at sites of DNA damage, which we propose may contribute to the presentation of DIADEM by compromising genome integrity and cell division during development.

4″-modified azithromycin derivatives bearing nitrogen-containing heterocycles with dual inhibitory activity.

Tribenzyltin carboxylates complexes, namely tri(4-fluorobenzyl)tin[(N,N-diisopropylcarbamothioyl)sulfanyl]acetate (C1) and tribenzyltin isonicotinate (C9), have been reported to exhibit significant anticancer activity via the death receptor and mitochondrial apoptotic pathway. However, understanding their upstream mechanism remains crucial. This study utilized both in silico and in vitro approaches to elucidate their mechanism of action. Molecular docking analysis indicated that C1 and C9 interacted with the minor groove of DNA. Fluorescence displacement assays confirmed their interaction with DNA through the minor groove. In enzyme inhibition assays, C1 showed significant inhibition of thioredoxin reductase (TrxR) activity, while C9 exhibited a dose-dependent effect, consistent with their docking affinities (- 4.1 and - 4.3kcal/mol, respectively). Both compounds strongly inhibited human DNA topoisomerase I (TopI), supported by docking energies of - 7.0 and - 6.6kcal/mol, respectively. The nearly complete inhibition of TopI across all treatment groups highlights it as a key molecular target. Additionally, C1 and C9 bound to thymidylate synthase (TS) with docking energies of - 7.2 and - 7.7kcal/mol, respectively, leading to a marked reduction in TS levels. Molecular dynamics simulations of the TopI, TS, and TrxR complexes revealed that both C1 and C9 maintained stable binding within their respective active sites throughout the 100ns trajectories. In summary, C1 and C9 primarily target the DNA minor groove and inhibit key enzymes such as TopI, TrxR, and TS, shedding light on their intricate mechanisms of action. These findings underscore the potential of C1 and C9 as promising multitarget metal-based anticancer agents.

This study aims to explore biomarkers linked to the progression from non-alcoholic fatty liver disease (NAFLD) to hepatocellular carcinoma (HCC) and their therapeutic potential. Using bioinformatics, we identified key differentially expressed genes from various databases, focusing on genes related to NAFLD, non-alcoholic steatohepatitis, liver cirrhosis, and HCC. Among the upregulated genes, baculoviral IAP repeat containing 5 (BIRC5), cyclin B1 (CCNB1), cyclin-dependent kinase 1 (CDK1), and DNA topoisomerase II alpha (TOP2A) were found to be significant. In vivo and in vitro models of NAFLD and clinical HCC samples validated BIRC5 as a critical regulator in the disease progression. Functional assays revealed that knocking down BIRC5 alleviated fatty acid-induced liver damage and mitochondrial dysfunction in NAFLD models, while also inhibiting HCC cells proliferation and migration, further leading to mitochondrial dysfunction. YM155 (a specific BIRC5 inhibitor) also confirmed the previous experimental results. Then we performed experiments using BIRC5 overexpression plasmid. BIRC5 overexpression exacerbated hepatic steatosis and mitochondrial function in free fatty acid (FFA) -induced AML12 hepatocytes, and enhanced HCC cells proliferation, migration, and invasion. These findings highlight BIRC5 as a pivotal driver in the NAFLD-HCC transition, mediating metabolic dysfunction and malignant transformation. This study proposes BIRC5 as a therapeutic target and diagnostic biomarker, offering perspectives for HCC diagnosis and treatment of HCC. These results underscore the importance of BIRC5 in halting NAFLD-HCC progression and provide valuable insights for future clinical applications.