Search Results (57)

Pancreatic ductal adenocarcinoma (PDAC) remains a highly lethal malignancy with few therapeutic options. Topoisomerase IIα (TOPO2α) is frequently overexpressed in PDAC and is associated with poor clinical outcomes, yet current TOPO2α-directed therapies are constrained by limited efficacy and toxicity. Barettin, a brominated indole-containing diketopiperazine isolated from the marine sponge Geodia barretti, has not previously been evaluated against PDAC-relevant targets. Here, we identify barettin as a TOPO2α inhibitor using an integrated phenotypic, computational, and biochemical approach. Barettin exerts a cytostatic, non-toxic effect, selectively suppressing proliferation in a subset of PDAC models while showing reduced activity in others, revealing context-dependent efficacy and biological selectivity. Consistent with this, barettin inhibits TOPO2α-mediated DNA decatenation in vitro, demonstrating direct interference with enzyme activity. These findings support barettin as a selective inhibitor of a cancer-relevant proliferative pathway, uncovering a potential vulnerability in a subset of PDAC. Full article

Cancer represents a major global health challenge, contributing to an estimated 19 million new cases annually. While conventional chemotherapeutic approaches continue to advance, target-based therapeutic strategies are increasingly recognized as effective pathways in modern drug development. A prominent biological target in current anticancer research is the selective inhibition of Topoisomerase II alpha (TOP2A). TOP2A, a crucial DNA topoisomerase, is vital for maintaining genomic integrity by mediating the cleavage and re-ligation of double-stranded DNA during essential cellular processes, such as DNA replication and transcription. Inhibiting TOP2A effectively disrupts these processes, leading to cell death. This study employed computer-aided drug design approaches to virtually screen a library of 3000 xanthone derivatives against the TOP2A target, and the results were preliminarily validated through cytotoxicity assays on the A549 and HepG2 cancer cell lines. The computational methods utilized included molecular docking, pharmacological modeling, molecular dynamics simulations, and steered molecular dynamics simulations. The virtual screening identified two highly promising HIT compounds, CID162372098 and CID156619937, that exhibited the most favorable interactions and stability profiles in relation to the TOP2A active site. The experimental results demonstrated that both hit compounds effectively exhibited significant anti-proliferative activities against the HepG2 cell line, with IC50 values of 9.54 ± 0.26 µg mL⁻¹ (CID162372098) and 10.03 ± 0.36 12.69 ± 0.31 µg mL⁻¹ (CID156619937), respectively. Collectively, these findings demonstrate the potential of xanthone-based scaffolds as inhibitors of TOP2A and provide a rational framework for the screening and development of novel anticancer agents. Full article

Background: Heterocycle compounds with acridine, quinoline, indole, and pyridine nuclei are potentially active for anticancer activity since they can promote inhibition of vital enzymes, decreasing cell survival after binding to biomolecules. However, unspecific biological interactions can result in unwanted effects, which should be defined during the synthesis and proposition of new molecules. Thus, the objective of this study was to investigate the biological and teratogenic effects of four nitrogen heterocycles proposed for anticancer therapy. Methods: Four 2-cyano-N-phenylacrylamine type derivatives containing acridine (3a), quinoline (3b), indole (3c), and pyridine (3d) nuclei were synthesized and characterized. They were evaluated for their ability to interact with DNA, physicochemical and pharmacokinetic predictions, in vitro and in silico methodologies, besides in vitro inhibition of the Topoisomerase IIα enzyme, antiproliferative activity in tumor and non-tumor cells, hemolytic activity with human erythrocytes, and in vivo toxicological studies with zebrafish embryos. Results: UV–vis absorption studies with ssDNA revealed different spectroscopic effects, with binding constants (Kb) ranging from 1.41 × 10⁵ to 6.46 × 10⁴ M⁻¹. The fluorescence quenching constant (Ksv) with ethidium bromide (EB) varied between 0.53 and 0.67 × 10³ M⁻¹. The compounds intercalated into DNA base pairs, a mechanism confirmed by molecular docking, with 3b (quinoline) showing the most substantial interaction. All derivatives exhibited antitopoisomerase IIα activity at 100 μM and were cytotoxic against MCF-7 and T47-D breast tumor cells, particularly against the more aggressive T47-D lineage. No hemolytic activity was observed in human erythrocytes. In vivo assays in zebrafish embryos showed no toxicological or cardiotoxic effects. However, all compounds altered superoxide dismutase (SOD) and catalase (CAT) enzymatic activity, requiring further studies on reactive oxygen species (ROS) generation to assess potential adverse effects. Furthermore, significant results were observed in the physicochemical and pharmacokinetic parameters of the synthesized compounds. Conclusions: The findings highlight the quinoline derivative (3b) as the most promising nitrogen heterocycle due to its antiproliferative activity and biomolecular interactions without adverse effects in zebrafish embryos, distinguishing it from clinically available agents. Full article

Cancer remains one of the leading causes of morbidity and mortality worldwide, demanding the continuous search for novel and more selective chemotherapeutic agents. Quinones, particularly naphthoquinones, constitute a privileged class of redox-active compounds with well-documented antitumor activity. Likewise, thiazoles represent a heterocyclic scaffold widely explored in medicinal chemistry due to their broad pharmacophoric adaptability and diverse biological activities. In this context, this review comprehensively explores the chemical synthesis and anticancer potential of hybrid molecules combining the naphthoquinone and thiazole scaffolds. The hybridization of these pharmacophores has emerged as a powerful strategy to design multitarget antitumor agents. The review summarizes key synthetic methodologies, including Hantzsch, hetero Diels–Alder cycloaddition and multicomponent reactions, leading to structurally diverse hybrids. Particular emphasis is placed on derivatives exhibiting strong cytotoxic effects against a broad spectrum of cancer cell lines (e.g., OVCAR3, MCF-7, A549, HCT-116, HeLa, and Jurkat), low toxicity toward normal cells and well-defined mechanisms of action involving topoisomerase IIα, EGFR, STAT3, and CDK1 inhibition, as well as ROS generation and cell cycle arrest. Among these, certain hybrids displayed nanomolar potency and high selectivity indices, reinforcing their potential as promising lead compounds for anticancer drug development. Full article

Background/Objectives: In the current medical era, Topoisomerase II is recognized as an essential enzyme that regulates DNA topology during critical biological processes such as DNA replication, transcription, and repair. This study aimed to design, synthesize, and biologically evaluate a new series of pyrazolo[3,4-b]pyridines (8a–g, 10a–g, and 12) as potential anticancer agents and Topoisomerase II inhibitors. Methods: The synthesized compounds were subjected to in vitro anticancer screening at the National Cancer Institute (NCI, USA). Active derivatives were further evaluated through a five-dose screening to determine their antiproliferative potency. Selected compounds were examined for their effects on leukemia cell lines (K562 and MV4-11), and mechanistic studies were performed to assess DNA damage, cell cycle distribution, and apoptosis-related protein modulation. Additionally, enzyme inhibition assays were conducted to determine Topoisomerase IIα (TOPIIα) inhibition. Results: Initial single-dose screening identified several active compounds, notably 8b, 8c, 8e, 8f, 10b, 10c, 10e, and 10f. Among these, compound 8c exhibited potent and broad-spectrum antiproliferative activity across the NCI cancer cell line panel, with a GI₅₀ MG-MID value of 1.33 µM (range: 0.54–2.08 µM). The synthesized molecules showed moderate to good anti-leukemic efficacy against K562 and MV4-11 cells. Mechanistic investigations revealed that compound 8c induced DNA damage and S-phase cell cycle arrest, leading to apoptosis as evidenced by the modulation of PARP-1, Bax, XIAP, and Caspases. Furthermore, target-based assays confirmed that compound 8c significantly inhibited the DNA relaxation activity of TOPIIα in a dose-dependent manner, comparable to etoposide. Conclusions: The study highlights compound 8c as a promising pyrazolo[3,4-b]pyridine derivative with potent antiproliferative activity and effective inhibition of Topoisomerase IIα. These findings suggest its potential as a lead scaffold for further optimization in anticancer drug development.. Full article

Background/Objectives: Intrinsically disordered protein regions (IDRs) are difficult to study due to their flexible nature and transient interactions. Computational folding using AlphaFold may offer one way to explore potential folding of these regions under various conditions. Human DNA topoisomerase IIα (TOP2A) is an essential enzyme involved in regulating DNA topology during replication and cell division. TOP2A has an IDR at the C-terminal domain (CTD) that has been shown to be important for regulating TOP2A function, but little is known about potential conformations that it may undertake. Methods: Utilizing the AlphaFold 3 (AF3) model by way of AlphaFold Server, TOP2A was folded as a dimer first without and then with 29 literature-supported post-translational modifications (PTMs) and DNA to observe whether there is predicted folding. Results: TOP2A CTD does not fold in the absence of PTMs. With the addition of PTMs, however, the CTD is predicted to fold into a globular bundle of loops and α-helices. While DNA alone did not induce folding, in the presence of PTMs, DNA ligands increased helicity of the folded CTD and caused it to interact at different core domain interfaces. In addition, DNA is predicted to enable folding of the TOP2A CTD in the presence of fewer PTMs when compared to the absence of DNA. Conclusions: AF3 predicts the folding of TOP2A CTD in the presence of specific PTMs, and this folding appears to shift to allow binding to DNA in functionally relevant regions. These studies provide predicted folding patterns that can be tested by biochemical approaches. AF3 may support the development of testable hypotheses regarding IDRs and enables researchers to model protein-DNA interactions. Full article

Background: 1,4-bis-L/L methionine–conjugated mitoxantrone–amino acid conjugate (L/LMet-MAC) inhibits topoisomerase IIα and enhances tumor cytotoxicity, but its short half-life limits therapeutic application. Objective: To improve the pharmacokinetics and antitumor efficacy of L/LMet-MAC through liposomal encapsulation. Methods: PEGylated DSPC liposomes containing EPG or prepared via the ammonium sulfate gradient method were employed to encapsulate L/LMet-MAC. Encapsulation efficiency, drug-to-lipid ratio, and serum stability were assessed. Pharmacokinetics, antitumor efficacy, and systemic safety were further evaluated in vivo. Results: L/LMet-MAC encapsulated in PEGylated DSPC liposomes containing EPG or prepared using the ammonium sulfate gradient method has high encapsulation efficiency. Further studies show that PEGylated DSPC liposomes prepared with the ammonium sulfate gradient approach display an efficient D/L ratio and serum stability as well as improved pharmacokinetics and enhanced antitumor efficacy while mitigating the side effects of L/LMet-MAC. Conclusions: PEGylated DSPC liposomes prepared using an ammonium sulfate gradient showed favorable performance for delivering L/LMet-MAC. Full article

Human topoisomerase IIα and IIβ regulate DNA topology and knots in chromosomes during crucial cellular processes, making these enzymes common targets for anticancer drugs. However, selective inhibition of topoisomerase IIα (TOP2A) is desired to decrease adverse effects, which may be mediated by topoisomerase IIβ (TOP2B). The main region of difference between the two isoforms is the intrinsically disordered C-terminal domain (CTD), which is being studied as a target for selective inhibition. Our previous work examined several regions within the CTD to determine whether those regions impact biochemical function. In this current study, we designed and constructed four TOP2A mutants with amino acid substitutions in the CTD, which were then assessed for impact on biochemical activity. V1482D exhibited increased levels of relaxation, while both V1482D and K1520I exhibited increased levels of decatenation. No major impact on DNA cleavage or binding were observed with any of the mutants. The isolated impact of the changes on relaxation and decatenation supports the concept that the CTD can affect one aspect of the enzyme’s function in an isolated manner, which was seen in our previous study. Taken together, these results suggest that modification of specific positions within the CTD affects substrate selection. These results are mapped onto the CTD for consideration of potential regions to target for inhibition of TOP2A. Full article

Compounds with thiosemicarbazide and semicarbazide scaffolds are among the most promising structures in medicinal chemistry due to the possibility of forming multiple hydrogen bonds. Therefore, six new derivatives of 4-fluorophenoxyacetylthiosemicarbazide and 4-fluorophenoxyacetylthiosemicarbazide were designed to compare their physicochemical properties, biological activity, and in silico pharmacokinetic parameters. All compounds were characterized by ¹H, ¹³C NMR, ¹⁹F, IR spectra. For selected derivatives (AB2 and AB5), X-ray studies were performed to confirm their synthetic route and identify the tautomeric forms and intra- and intermolecular interactions occurring in the crystalline state. In the in silico pharmacokinetic study, a clear difference in lipophilicity was observed between thiosemicarbazide and semicarbazide derivatives. In vitro biological studies have shown the promising activity of thiosemicarbazides against prostate cancer cell line LNCaP, with a higher safety profile than semicarbazides. The most active compound AB2 showed IC₅₀ = 108.14 μM against LNCaP. Based on biological studies, topoisomerase IIα was proposed as a potential molecular target, which was confirmed by molecular docking studies. Full article

Artemisia absinthium L., is a plant with established pharmacological properties, but the A. absinthium root extract (AARE) remains unexplored. The aim of this study was to examine the chemical composition of AARE and assess its biological activity, which included antidiabetic, antibacterial, anticancer, and antioxidant properties. GC-MS was used to analyze the chemical components. The antioxidant activity of the total phenolic and flavonoid content was evaluated. Antibacterial activity and cytotoxic effects were identified. Enzyme inhibition experiments were performed to determine its antidiabetic potential. Molecular docking was utilized to evaluate the potential antioxidant, antibacterial, and anticancer activities of the compounds from AARE using Maestro 11.5 from the Schrödinger suite. AARE exhibited moderate antioxidant activity in DPPH (IC₅₀: 172.41 ± 3.15 μg/mL) and ABTS (IC₅₀: 378.94 ± 2.18 μg/mL) assays. Cytotoxicity tests on MCF-7 and HepG2 cancer cells demonstrated significant anticancer effects, with IC₅₀ values of 150.12 ± 0.74 μg/mL and 137.11 ± 1.33 μg/mL, respectively. Apoptotic studies indicated an upregulation of pro-apoptotic genes (caspase-3, 8, 9, Bax) and a downregulation of anti-apoptotic markers (Bcl-2 and Bcl-Xl). AARE also inhibited α-amylase and α-glucosidase, suggesting potential antidiabetic effects, with IC₅₀ values of 224.12 ± 1.17 μg/mL and 243.35 ± 1.51 μg/mL. Antibacterial assays revealed strong activity against Gram-positive bacteria. Molecular docking and pharmacokinetic analysis identified promising inhibitory effects of key AARE compounds on NADPH oxidase, E. coli Gyrase B, and Topoisomerase IIα, with favorable drug-like properties. These findings suggest AARE’s potential in treating cancer, diabetes, and bacterial infections, warranting further in vivo and clinical studies. Full article

Organometallic complexes of fac-tricarbonylrhenium have been shown to exhibit anticancer properties. Anthrapyrazole anticancer agents act as DNA intercalators and topoisomerase IIα inhibitors, leading to double-strand breaks (DBS) and cell cycle arrest. This work involves the synthesis and biological evaluation of novel fac-tricarbonyl-rhenium complexes with anthrapyrazole derivatives. The anthrapyrazole moiety was synthesized from 1,8-dihydroxyanthraquinone, and three ligands L1, L2 and L3 were prepared. Ligand L1 coordinates via the phenolic O and pyrazole N as bidentate chelator forming the fac-[Re(CO)₃(κ²-N,O)(MeOH)]-type complex, ReL1. Ligand L2 contains a pendant picolylamine N,N′-chelating system, forming the bidentate fac-[Re(CO)₃(κ²-N,N′)Br]-type complex, ReL2. Ligand L3 contains a pendant picolylaminomonoacetic acid chelating system, forming a tridentate fac-[Re(CO)₃(κ³-N,N′,O)]-type complex, ReL3. Complex ReL4 contains a picolylamine chelator, forming a complex with structure fac-[Re(CO)₃(κ²-N,N′)Br], which was synthesized as a model for ReL2, and its coordination mode was resolved by X-ray crystallography. The complexes were characterized spectroscopically, and their biological properties were evaluated in vitro, in terms of DNA binding as well as for the cytotoxicity against CT-26 tumor cell line. Tumor cell cytotoxicity was high for ligand L2 and complex ReL2, exhibiting IC₅₀ values of 0.36 and 0.64 μΜ, respectively. The most promising complex ReL2 was evaluated further by the preparation of its congener γ-emitting technetium-99m radio-complex, ^99mTcL2. The in vitro uptake in CT26 tumor cells and the in vivo uptake in CT26 tumor-bearing mice of ^99mTcL2 was determined, and its pharmacokinetic profile was established. These data indicate that the ^99mTc complex has suitable properties to enter tumor cells in vitro and in vivo, and therefore ReL2 is promising for further evaluation. Full article

DNA Topoisomerase IIα (Top2A) is a nuclear enzyme that is a cancer drug target, and there is interest in identifying novel sites on the enzyme to inhibit cancer cells more selectively and to reduce off-target toxicity. The C-terminal domain (CTD) is one potential target, but it is an intrinsically disordered domain, which prevents structural analysis. Therefore, we set out to analyze the sequence of Top2A from 105 species using bioinformatic analysis, including the PSICalc algorithm, Shannon entropy analysis, and other approaches. Our results demonstrate that large (10th-order) interdependent clusters are found including non-proximal positions across the major domains of Top2A. Further, CTD-specific clusters of the third, fourth, and fifth order, including positions that had been previously analyzed via mutation and biochemical assays, were identified. Some of these clusters coincided with positions that, when mutated, either increased or decreased relaxation activity. Finally, sites of low Shannon entropy (i.e., low variation in amino acids at a given site) were identified and mapped as key positions in the CTD. Included in the low-entropy sites are phosphorylation sites and charged positions. Together, these results help to build a clearer picture of the critical positions in the CTD and provide potential sites/regions for further analysis. Full article

Breast cancer is currently the most diagnosed form of cancer and the leading cause of death by cancer among females worldwide. We described the family of long non-coding mitochondrial RNAs (ncmtRNAs), comprised of sense (SncmtRNA) and antisense (ASncmtRNA) members. Knockdown of ASncmtRNAs using antisense oligonucleotides (ASOs) induces proliferative arrest and apoptotic death of tumor cells, but not normal cells, from various tissue origins. In order to study the mechanisms underlying this selectivity, in this study we performed RNAseq in MDA-MB-231 breast cancer cells transfected with ASncmtRNA-specific ASO or control-ASO, or left untransfected. Bioinformatic analysis yielded several differentially expressed cell-cycle-related genes, from which we selected Aurora kinase A (AURKA) and topoisomerase IIα (TOP2A) for RT-qPCR and western blot validation in MDA-MB-231 and MCF7 breast cancer cells, as well as normal breast epithelial cells (HMEC). We observed no clear differences regarding mRNA levels but both proteins were downregulated in tumor cells and upregulated in normal cells. Since these proteins play a role in genomic integrity, this inverse effect of ASncmtRNA knockdown could account for tumor cell downfall whilst protecting normal cells, suggesting this approach could be used for genomic protection under cancer treatment regimens or other scenarios. Full article

The dynamic processes operating on genomic DNA, such as gene expression and cellular division, lead inexorably to topological challenges in the form of entanglements, catenanes, knots, “bubbles”, R-loops, and other outcomes of supercoiling and helical disruption. The resolution of toxic topological stress is the function attributed to DNA topoisomerases. A prominent example is the negative supercoiling (nsc) trailing processive enzymes such as DNA and RNA polymerases. The multiple equilibrium states that nscDNA can adopt by redistribution of helical twist and writhe include the left-handed double-helical conformation known as Z-DNA. Thirty years ago, one of our labs isolated a protein from Drosophila cells and embryos with a 100-fold greater affinity for Z-DNA than for B-DNA, and identified it as topoisomerase II (gene Top2, orthologous to the human UniProt proteins TOP2A and TOP2B). GTP increased the affinity and selectivity for Z-DNA even further and also led to inhibition of the isomerase enzymatic activity. An allosteric mechanism was proposed, in which topoII acts as a Z-DNA-binding protein (ZBP) to stabilize given states of topological (sub)domains and associated multiprotein complexes. We have now explored this possibility by comprehensive bioinformatic analyses of the available protein sequences of topoII representing organisms covering the whole tree of life. Multiple alignment of these sequences revealed an extremely high level of evolutionary conservation, including a winged-helix protein segment, here denoted as Zτ, constituting the putative structural homolog of Zα, the canonical Z-DNA/Z-RNA binding domain previously identified in the interferon-inducible RNA Adenosine-to-Inosine-editing deaminase, ADAR1p150. In contrast to Zα, which is separate from the protein segment responsible for catalysis, Zτ encompasses the active site tyrosine of topoII; a GTP-binding site and a GxxG sequence motif are in close proximity. Quantitative Zτ-Zα similarity comparisons and molecular docking with interaction scoring further supported the “B-Z-topoII hypothesis” and has led to an expanded mechanism for topoII function incorporating the recognition of Z-DNA segments (“Z-flipons”) as an inherent and essential element. We further propose that the two Zτ domains of the topoII homodimer exhibit a single-turnover “conformase” activity on given G(ate) B-DNA segments (“Z-flipins”), inducing their transition to the left-handed Z-conformation. Inasmuch as the topoII-Z-DNA complexes are isomerase inactive, we infer that they fulfill important structural roles in key processes such as mitosis. Topoisomerases are preeminent targets of anti-cancer drug discovery, and we anticipate that detailed elucidation of their structural–functional interactions with Z-DNA and GTP will facilitate the design of novel, more potent and selective anti-cancer chemotherapeutic agents. Full article

In 2020, breast cancer became the most frequently diagnosed type of cancer, with nearly 2.3 million new cases diagnosed. However, with early diagnosis and proper treatment, breast cancer has a good prognosis. Here, we investigated the effect of thiosemicarbazide derivatives, previously identified as dual inhibitors targeting topoisomerase IIα and indoleamine-2,3-dioxygenase 1 (IDO 1), on two distinct types of breast cancer cells (MCF-7 and MDA-MB-231). The investigated compounds (1–3) selectively suppressed the growth of breast cancer cells and promoted apoptosis via caspase-8- and caspase-9-related pathways. Moreover, these compounds caused S-phase cell cycle arrest and dose-dependently inhibited the activity of ATP-binding cassette transporters (MDR1, MRP1/2 and BCRP) in MCF-7 and MDA-MB-231 cells. Additionally, following incubation with compound 1, an increased number of autophagic cells within both types of the investigated breast cancer cells was observed. During preliminary testing of ADME-Tox properties, the possible hemolytic activities of compounds 1–3 and their effects on specific cytochrome P450 enzymes were evaluated. Full article