DNA Relaxation Research Articles

A high throughput single molecule platform to study DNA supercoiling effect on protein-DNA interactions.

DNA supercoiling significantly influences DNA metabolic pathways. To examine its impact on DNA-protein interactions at the single-molecule level, we developed a highly efficient and reliable protocol to modify plasmid DNA at specific sites, allowing us to label plasmids with fluorophores and biotin. We then induced negative and positive supercoiling in these plasmids using gyrase and reverse gyrase, respectively. Comparing supercoiled DNA with relaxed circular DNA, we assessed the effects of supercoiling on CRISPR-Cas9 and mismatch repair protein MutS. We found that negative DNA supercoiling exacerbates off-target effects in DNA unwinding by Cas9. For MutS, we observed both negative and positive DNA supercoiling enhances the binding interaction between MutS and a mismatched base pair but does not affect the rate of ATP-induced sliding clamp formation. These findings not only underscore the versatility of our protocol but also opens new avenues for exploring the intricate dynamics of protein-DNA interactions under the influences of supercoiling.

Flow dichroism of DNA can be quantitatively predicted via coarse-grained molecular simulations

We demonstrate the use of multiscale polymer modeling to quantitatively predict DNA linear dichroism (LD) in shear flow. LD is the difference in absorption of light polarized along two perpendicular axes and has long been applied to study biopolymer structure and drug-biopolymer interactions. As LD is orientation dependent, the sample must be aligned in order to measure a signal. Shear flow via a Couette cell can generate the required orientation; however, it is challenging to separate the LD due to changes in polymer conformation from specific interactions, e.g., drug-biopolymer. In this study, we have applied a combination of Brownian dynamics and equilibrium Monte Carlo simulations to accurately predict polymer alignment, and hence flow LD, at modest computational cost. As the optical and conformational contributions to the LD can be explicitly separated, our findings allow for enhanced quantitative interpretation of LD spectra through the use of an in silico model to capture conformational changes. Our model requires no fitting and only five input parameters: the DNA contour length, persistence length, optical factor, solvent quality, and relaxation time, all of which have been well characterized in prior literature. The method is sufficiently general to apply to a wide range of biopolymers beyond DNA, and our findings could help guide the search for new pharmaceutical drug targets via flow LD.

Inhibition of DNA Topoisomerase Ι by Flavonoids and Polyacetylenes Isolated from Bidens pilosa L.

Human DNA topoisomerase I (Topo I) is an essential enzyme in regulating DNA supercoiling during transcription and replication, and it is an important therapeutic target for anti-tumor agents. Bidens pilosa L. is a medicinal herb that is used as a folk medicine for cancers in China. A new flavonoid (1) and a new polyacetylene (20), along with eighteen flavonoids (2-19) and nine polyacetylenes (21-29), were isolated and identified from the methanol extract of the whole plant of B. pilosa, and some of the compounds (4, 5, 6 and 7) exhibited potent cytotoxicity against a panel of five human cancer cell lines. The DNA relaxation assay revealed that some flavonoids and polyacetylenes exerted inhibitory activities on human DNA Topo I, among them compounds 1, 2, 5, 6, 7, 8, 15, 19, 20, 22, and 24 were the most active ones, with IC50 values of 393.5, 328.98, 145.57, 239.27, 224.38, 189.84, 89.91, 47.5, 301.32, 178.03, and 218.27 μM, respectively. The structure-activity analysis of flavonoids was performed according to the results from the Topo I inhibition assay. The DNA content analysis revealed that 5, 6, and 7 potently arrested cell cycle at the G1/S and G2/M phases in human colon cancer cell DLD-1 depending on the concentration of the inhibitors. The levels of protein expression related to the G1/S and G2/M cell cycle checkpoints were in accordance with the results from the DNA content analysis. These findings suggest that flavonoids are one of the key active ingredients accounting for the anti-tumor effect of B. pilosa.

From Bioreactor to Bulk Rheology: Achieving Scalable Production of Highly Concentrated Circular DNA.

DNA serves as a model system in polymer physics due to its ability to be obtained as a uniform polymer with controllable topology and nonequilibrium behavior. Currently, a major obstacle in the widespread adoption of DNA is obtaining it on a scale and cost basis that accommodates bulk rheology and high-throughput screening. To address this, recent advancements in bioreactor-based plasmid DNA production is coupled with anion exchange chromatography producing a unified approach to generating gram-scale quantities of monodisperse DNA. With this method, 1.1 grams of DNA is obtained per batch to generate solutions with concentrations up to 116mg mL-1. This solution of uniform supercoiled and relaxed circular plasmid DNA, is roughly 69 times greater than the overlap concentration. The utility of this method is demonstrated by performing bulk rheology measurements at sample volumes up to 1 mL on DNA of different lengths, topologies, and concentrations. The measured elastic moduli are orders of magnitude larger than those previously reported for DNA and allowed for the construction of a time-concentration superposition curve that spans 12 decades of frequency. Ultimately, these results can provide important insights into the dynamics of ring polymers and the nature of highly condensed DNA dynamics.

Mechanisms of the Antineoplastic Effects of New Fluoroquinolones in 2D and 3D Human Breast and Bladder Cancer Cell Lines.

Antibacterial fluoroquinolones have emerged as potential anticancer drugs, thus prompting the synthesis of novel molecules with improved cytotoxic characteristics. Ciprofloxacin and norfloxacin derivatives, previously synthesized by our group, showed higher anticancer potency than their progenitors. However, no information about their mechanisms of action was reported. In this study, we selected the most active among these promising molecules and evaluated, on a panel of breast (including those triple-negative) and bladder cancer cell lines, their ability to induce cell cycle alterations and apoptotic and necrotic cell death through cytofluorimetric studies. Furthermore, inhibitory effects on cellular migration, metalloproteinase, and/or acetylated histone protein levels were also evaluated by the scratch/wound healing assay and Western blot analyses, respectively. Finally, the DNA relaxation assay was performed to confirm topoisomerase inhibition. Our results indicate that the highest potency previously observed for the derivatives could be related to their ability to induce G2/M cell cycle arrest and apoptotic and/or necrotic cell death. Moreover, they inhibited cellular migration, probably by reducing metalloproteinase levels and histone deacetylases. Finally, topoisomerase inhibition, previously observed in silico, was confirmed. In conclusion, structural modifications of progenitor fluoroquinolones resulted in potent anticancer derivatives possessing multiple mechanisms of action, potentially exploitable for the treatment of aggressive/resistant cancers.

Ratiometric nonfluorescent CRISPR assay utilizing Cas12a-induced plasmid supercoil relaxation

Most CRISPR-based biosensors rely on labeled reporter molecules and expensive equipment for signal readout. A recent approach quantifies analyte concentration by sizing λ DNA reporters via gel electrophoresis, providing a simple solution for label-free detection. Here, we report an alternative strategy for label-free CRISPR-Cas12a, which relies on Cas12a trans-nicking induced supercoil relaxation of dsDNA plasmid reporters to generate a robust and ratiometric readout. The ratiometric CRISPR (rCRISPR) measures the relative percentage of supercoiled plasmid DNA to the relaxed circular DNA by gel electrophoresis for more accurate target concentration quantification. This simple method is two orders of magnitude more sensitive than the typical fluorescent reporter. This self-referenced strategy solves the potential application limitations of previously demonstrated DNA sizing-based CRISPR-Dx without compromising the sensitivity. Finally, we demonstrated the applicability of rCRISPR for detecting various model DNA targets such as HPV 16 and real AAV samples, highlighting its feasibility for point-of-care CRISPR-Dx applications.

Nuclear-penetrating scleroderma autoantibody inhibits topoisomerase 1 cleavage complex formation

The contributions of anti-Topoisomerase 1 (Top1) autoantibodies to the pathophysiology of diffuse cutaneous systemic sclerosis (dcSSc), the most aggressive scleroderma subtype, are unknown. Top1 catalyzes DNA relaxation and unwinding in cell nuclei, a site previously considered inaccessible to antibodies. The discovery of autoantibodies in systemic lupus erythematosus that penetrate nuclei and inhibit DNA repair raised the possibility that nuclear-penetrating autoantibodies contribute to mechanisms of autoimmunity. Here we show that an anti-Top1 autoantibody produced by a single B cell clone from a patient with dcSSc penetrates live cells and localizes into nuclei. Functionally, the autoantibody inhibits formation of the Top1 cleavage complex necessary for DNA nicking, which distinguishes it from cytotoxic camptothecin Top1 inhibitors used in cancer therapy that trap the cleavage complex rather than preventing its formation. Discovery of a patient-derived cell-penetrating scleroderma anti-Top1 autoantibody that inhibits Top1 cleavage complex formation supports the hypothesis that anti-Top1 autoantibodies contribute to cellular dysfunction in dcSSc and offers a valuable antibody reagent resource for future studies on anti-Top1 autoantibody contributions to scleroderma pathophysiology.

Activation of the cGAS-STING pathway by viral dsDNA leading to M1 polarization of macrophages mediates antiviral activity against hepatitis B virus

Background and aimsActivation of the cGAS-STING pathway induces the production of type I interferons, initiating the antiviral immune response, which contributes to the clearance of pathogens. Previous studies have shown that STING agonists promote hepatitis B virus (HBV) clearance; however, few studies have investigated the effect of activating the cGAS-STING pathway in macrophages on HBV. MethodsThe polarization status of HBV particle-stimulated RAW264.7 macrophages was analyzed. After stimulation with HBV particles, the analysis focused on determining whether the DNA sensors in RAW264.7 macrophages recognized the viral double-stranded DNA (dsDNA) and evaluating the activation of the cGAS-STING pathway. Coculture of mouse macrophages and hepatocytes harboring HBV was used to study the antiviral activity of HBV-stimulated RAW264.7 macrophages. ResultsAfter stimulation with HBV particles, HBV relaxed circular DNA (rcDNA) was detected in RAW264.7 macrophages, and the protein expression of phospho-STING, phospho-TBK1, and phospho-IRF3 in the STING pathway was increased, as shown by Western blot analysis, which revealed that M1 polarization of macrophages was caused by increased expression of CD86. RT–PCR analyses revealed elevated expression of M1 macrophage polarization-associated cytokines such as TNFα, IL-1β, iNOS, and IFNα/β. In the coculture experiment, both HBsAg and HBeAg expression levels were significantly decreased in AML12-HBV1.3 cells cocultured with the supernatants of HBV-stimulated RAW264.7 macrophages. ConclusionThe results suggest that macrophages can endocytose HBV particles. Additionally, viral dsDNA can be recognized by DNA pattern recognition receptors, which in turn activate the cGAS-STING pathway, promoting the M1 polarization of macrophages, while no significant M2 polarization is observed. Macrophages stimulated with HBV particles exhibit enhanced antiviral activity against HBV.

Mechanisms of Hepatitis B Virus cccDNA and Minichromosome Formation and HBV Gene Transcription.

Hepatitis B virus (HBV) is the etiologic agent of chronic hepatitis B, which puts at least 300 million patients at risk of developing fibrosis, cirrhosis, and hepatocellular carcinoma. HBV is a partially double-stranded DNA virus of the Hepadnaviridae family. While HBV was discovered more than 50 years ago, many aspects of its replicative cycle remain incompletely understood. Central to HBV persistence is the formation of covalently closed circular DNA (cccDNA) from the incoming relaxed circular DNA (rcDNA) genome. cccDNA persists as a chromatinized minichromosome and is the major template for HBV gene transcription. Here, we review how cccDNA and the viral minichromosome are formed and how viral gene transcription is regulated and highlight open questions in this area of research.

Uniform extension–torsion of helical birods

An analytical formulation is presented to study uniform extension–torsion behavior of helical birods. The uniformity in extension and compression reduces the set of governing nonlinear differential equations of birod theory to a set of nonlinear algebraic equations for the relevant unknown variables of a birod. Upon normalizing the algebraic equations, we note that the birod’s coupled extension–torsion behavior in its natural state can be tuned by changing the birod’s constituent strand’s intrinsic curvature and twist, Poisson’s ratio and the birod’s stiffness parameters corresponding to inter-strand separation and rotation. Analytical expressions of the birod’s extensional stiffness, torsional stiffness, extension–torsion coupling stiffness, effective Poisson’s ratio, overwinding/unwinding effect etc. in the birod’s natural state are obtained. In particular, we show that depending on the parameters chosen, a torsionally relaxed birod can undergo non-intuitive slow overwinding followed by rapid unwinding during its stretching as observed during pulling of a torsionally relaxed DNA.

Structural basis of DNA crossover capture by Escherichia coli DNA gyrase.

DNA supercoiling must be precisely regulated by topoisomerases to prevent DNA entanglement. The interaction of type IIA DNA topoisomerases with two DNA molecules, enabling the transport of one duplex through the transient double-stranded break of the other, remains elusive owing to structures derived solely from single linear duplex DNAs lacking topological constraints. Using cryo-electron microscopy, we solved the structure of Escherichia coli DNA gyrase bound to a negatively supercoiled minicircle DNA. We show how DNA gyrase captures a DNA crossover, revealing both conserved molecular grooves that accommodate the DNA helices. Together with molecular tweezer experiments, the structure shows that the DNA crossover is of positive chirality, reconciling the binding step of gyrase-mediated DNA relaxation and supercoiling in a single structure.

Abstract LB180: Non-cardiotoxic properties of annamycin, a clinically evaluated anthracycline and potent topoisomerase 2β poison

Abstract Introduction. Cardiotoxicity is an important side effect limiting the clinical use of all anthracyclines, including doxorubicin (DOX). DOX targets topoisomerase II alpha (Top2α) and beta (Top2β), two enzymes that regulate DNA topology. Top2β is present in cardiomyocytes and has been proposed as a major contributor to DOX-induced cardiotoxicity. Annamycin (ANN), a novel clinically evaluated DOX analog, displays suppressed or no cardiotoxic properties in preclinical in vivo experiments. The objective of this study was to directly assess and compare the potency of DOX and ANN against Top2α and Top2β and determine their impact on physiology of human cardiomyocytes and mice hearts following chronic exposure. Our initial data are clearly aligned with lack of drug-related cardiotoxic events in ANN-treated patients in ongoing clinical trials. Methods. The potency of ANN and DOX to inhibit Top2 was tested in DNA relaxation assays using recombinant Top2α and Top2β with kinetoplast as the DNA substrate. ANN and DOX cytotoxicity was assessed in a panel of cancer cell lines cardiomyocytes (murine and human). In addition, the effects on cardiomyocyte physiology (beating rate, contraction, electric potential,) were assessed in human cardiomyocytes using the xCELLigence RTCA CardioECR. The pathophysiology of the heart after chronic exposure to the drugs was also evaluated in mice models. Cardiotoxicity of liposome formulated ANN (L-ANN) is currently being examined in ongoing clinical studies of subjects with acute myeloid leukemia (NCT05319587) or soft tissue sarcoma with metastases to the lungs (NCT04887298). Results. The nanomolar range potency of ANN in tested cancer cell lines was 2-10 times greater than that of DOX. The DNA relaxation assay demonstrated high potency of ANN against recombinant Top2α and Topβ with IC50 values 0.23±0.06 and 0.22± 0.06 µM, respectively, whereas the IC50 for DOX was 0.65± 0.22 and 0.34± 0.31µM, respectively. Importantly, ANN did not significantly affect electric potential, contractility, and viability of human cardiomyocytes up to highest tested dose of 1,500 nM (72h exposure). In contrast, DOX caused a dramatic change in heart beating rate and decreased in contractility and field potential amplitude. In rat cardiomyocyte culture (H92C), ANN was 14-fold less cytotoxic than DOX (IC50 1,264 nM for ANN vs. 88 nM for DOX). No cardiotoxicity of L-ANN was detected in myocardium of nude mice exposed weekly for 12 weeks to therapeutic doses of L-ANN (4 mg/kg, 40% of LD10). Finally, data from ongoing clinical trials revealed no drug related cardiac events in patients receiving L-ANN (confirmed by an independent review of the Duke’s ECHO core laboratory, USA). Conclusions. Our preclinical studies demonstrate non-cardiotoxic properties of ANN. These data are clearly aligned with lack of drug-related cardiotoxic events in ANN-treated patients in ongoing clinical trials. Interestingly, ANN appeared to be a significantly more potent inhibitor of Top2β than DOX, suggesting that Top2β may not be a universal mediator of cardiotoxicity of anthracyclines. Additional studies are needed to reevaluate the role of Top2β in anthracycline-induced cardiotoxicity. Citation Format: Rafal J. Zielinski, Krzysztof Grela, Roberto Cardenas-Zuniga, Stanislaw Skora, Izabela Fokt, Mihai Gagea, Wolfram Dempke, Waldemar Priebe. Non-cardiotoxic properties of annamycin, a clinically evaluated anthracycline and potent topoisomerase 2β poison [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB180.

Abstract 3367: R-loop type determines the orientation of transcription-replication conflicts attenuated by transcription factor IIS

Abstract During human Topoisomerase I (Top1) catalytic activity, a transient Top1-DNA cleavage complex (Top1cc) forms to allow DNA relaxation. Top1ccs can be stabilized by antitumor Top1 poisons, such as clinically-effective camptothecin analogs. Camptothecin-stabilized Top1ccs can lead to R-loop-dependent transcription-replication conflicts (TRCs), single-end DNA double-strand breaks (seDSBs) and genome instability in cancer cells. To define the genomic location of TRCs and the underlying mechanisms, we integrated genomic maps of Top1cc-induced DNA cleavage by END-seq and DNA:RNA hybrids by DRIP, determined the cell-cycle specificity of micronuclei formation and the effects of elongation transcription factor IIS (TFIIS). We have defined the genomic sites of both seDSBs and increased R-loop levels (gains) induced by Top1ccs. seDSBs are highly enriched at regions with R-loop gains at highly-transcribed genes in early replicating zones. Kinetics analyses of R-loops and genomic ChIP data of RNA polymerase II (RNAPII) revealed that R-loop gains can either be upstream or downstream of RNAPII. TFIIS effects showed that downstream R-loop gains can follow RNAPII backtracking and arrest. seDSB strandness showed that co-directional TRCs occur mainly at downstream hybrid-associated RNAPII, whereas head-on TRCs occur mainly at upstream hybrid-associated RNAPII. Therefore, Top1ccs can increase two structurally-different R-loops and R-loop-associated backtracked RNAPII causes co-directional TRCs at higher rate than head-on collisions. Moreover, Top1ccs trigger micronuclei formation at G1/early S, but not late S/G2, which are attenuated by TFIIS. Thus, our findings establish a role of RNAPII backtracking at highly-transcribed genes in Top1cc-induced co-directional TRCs and micronuclei formation. The findings can accelerate the development of new effective strategies to treat cancer patients. (Supported by Associazione Italiana per la Ricerca sul Cancro, Milan, Italy - IG 2019 - ID. 23032 to C.G. and by the European Union - NextGenerationEU through the Italian Ministry of University and Research under PNRR - M4C2-I1.3 Project PE_00000019 "HEAL ITALIA" to G.C., CUP J33C22002920006). Citation Format: Renée C. Duardo, Jessica Marinello, Marco Russo, Sara Morelli, Simona Pepe, Federico Guerra, Belen Gomez-González, Andrés Aguilera, Giovanni Capranico. R-loop type determines the orientation of transcription-replication conflicts attenuated by transcription factor IIS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3367.

Synthesis, Characterization, Docking Studies and in vitro Response of New Camptothecin Derivatives towards Oral Squamous Cell Carcinoma

Evidence suggests heterocyclic rings as the essential component of various available chemotherapeutics. Present study was aimed to carry out the molecular docking, synthesis, characterization and response of new camptothecin derivatives (NCDs) towards OSCC cell lines. To achieve the aim of the study, new camptothecin derivatives were designed to perform molecular docking against the target protein Human DNA Topoisomerase-1 (1T8I). Molecules 2 and 3 with high docking scores were subjected to synthesis. In current investigation, NCDs were synthesized by cyclization of imino analogue (2) into a new azetidinone derivative (3) on treatment with triethylamine and chloroacetyl chloride. Synthesized NCDs were characterized using IR, NMR and mass analysis. The NCDs were further subjected to antiproliferation study using CAL-27 (OSCC), followed by in vitro DNA relaxation assay and cell cycle analysis. The results of the docking of CPT-11 against Human DNA Topoisomerase-1 Duplex (PDB ID: 1T8I) in present study revealed compound 2 and 3 exhibited high docking score among all camptothecin analogues. Present study successfully synthesized and elucidated the structures of NCD 2 and 3. The antiproliferation study results revealed that NCD 2 and NCD 3 offered an IC50 of 34.73 µg/mL and 62.5 µg/mL, respectively. The DNA relaxation assay exhibited the inhibition action of synthesized NCDs (IC50 concentration) against topoisomerase enzyme. Moreover, the cell cycle analysis revealed that both NCDs arrested cancer cells in ‘S’ phase. Though the present study highlights the potential of NCDs against oral squamous cell carcinoma, however, the present study also recommends that the synthesized NCDs must be further evaluated for preclinical and clinical significance.

Structure-function analysis of the ATPase domain of African swine fever virus topoisomerase.

The ATPase domain of type II topoisomerase provides energy by hydrolyzing ATP and coordinates with the DNA-binding/cleavage domain to drive and control DNA transport. The precise molecular mechanisms of how these domains respond to DNA binding and ATP hydrolysis signals and communicate with each other remain elusive. We determine the first high-resolution crystal structure of the ATPase domain of African swine fever virus (ASFV) topo II in complex with AMPPNP and biochemically investigate its function in ATPase and DNA relaxation activities. Importantly, we find that mutations at three characteristic regions of the ASFV ATPase domain produce parallel effects on the basal/DNA-stimulated ATPase and relaxation activities, implying the tight coupling of the ATP hydrolysis and strand passage process. Therefore, our data provide important implications for understanding the strand passage mechanism of the type II topoisomerase and the structural basis for developing ATPase domain-targeting antivirals against ASFV.

The acidic C-terminal tail of DNA Gyrase of Salmonella enterica serovar Typhi controls DNA relaxation in an acidic environment

The intracellular bacteria, Salmonella Typhi adapts to acidic conditions in the host cell by resetting the chromosomal DNA topology majorly controlled by DNA Gyrase, a Type II topoisomerase. DNA Gyrase forms a heterodimer A2B2 complex, which manages the DNA supercoiling and relaxation in the cell. DNA relaxation forms a part of the regulatory mechanism to activate the transcription of genes required to survive under hostile conditions. Acid-induced stress attenuates the supercoiling activity of the DNA Gyrase, resulting in DNA relaxation. Salmonella DNA becomes relaxed as the bacteria adapt to the acidified intracellular environment. Despite comprehensive studies on DNA Gyrase, the mechanism to control supercoiling activity needs to be better understood. A loss in supercoiling activity in E. coli was observed upon deletion of the non-conserved acidic C-tail of Gyrase A subunit. Salmonella Gyrase also contains an acidic tail at the C-terminus of Gyrase A, where its deletion resulted in reduced supercoiling activity compared to wild-type Gyrase. Interestingly, we also found that wild-type Gyrase compromises supercoiling activity at acidic pH 2–3, thereby causing DNA relaxation. The absence of a C-tail displayed DNA supercoiling to some extent between pH 2–9. Hence, the C-tail of Gyrase A might be one of the controlling factors that cause DNA relaxation in Salmonella at acidic pH conditions. We propose that the presence of the C-tail of GyraseA causes acid-mediated inhibition of the negative supercoiling activity of Gyrase, resulting in relaxed DNA that attracts DNA-binding proteins for controlling the transcriptional response.

Dicobalt(ii) helices kill colon cancer cells via enantiomer-specific mechanisms; DNA damage or microtubule disruption.

Highly diastereoselective self-assembly reactions give both enantiomers (Λ and Δ) of anti-parallel triple-stranded bimetallic Co(ii) and Co(iii) cationic helices, without the need for resolution; the first such reaction for Co. The complexes are water soluble and stable, even in the case of Co(ii). Studies in a range of cancer and healthy cell lines indicate high activity and selectivity, and substantial differences between enantiomers. The oxidation state has little effect, and correspondingly, Co(iii) compounds are reduced to Co(ii) e.g. by glutathione. In HCT116 colon cancer cells the Λ enantiomer induces dose-dependent G2-M arrest in the cell cycle and disrupts microtubule architectures. This Co(ii) Λ enantiomer is ca. five times more potent than the isostructural Fe(ii) compound. Since the measured cellular uptakes are similar this implies a higher affinity of the Co system for the intracellular target(s); while the two systems are isostructural they have substantially different charge distributions as shown by calculated hydrophobicity maps. In contrast to the Λ enantiomer, Δ-Co(ii) induces G1 arrest in HCT116 cells, efficiently inhibits the topoisomerase I-catalyzed relaxation of supercoiled plasmid DNA, and, unlike the isostructural Fe(ii) system, causes DNA damage. It thus seems very likely that redox chemistry plays a role in the latter.

Metachromin C, a marine-derived natural compound, shows potential in antitumor activity.

Metachromin C was first isolated from the marine sponge Hippospongia metachromia and has been reported to possess potent cytotoxicity against leukemia cells. However, its antitumor activity and possible mechanisms in pancreatic cancer remain unclear. The effects of Metachromin C on cell viability were estimated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The compound demonstrated a cytotoxic effect on four pancreatic cancer cell lines (PANC-1, BxPC-3, MiaPaCa-2, and AsPC-1). The significant S phase arrest observed with Metachromin C treatment suggests its impact on DNA replication machinery. Metachromin C might interfere with the binding of Topoisomerase I (TOPO I) to DNA, inhibit TOPO I activity, prevent DNA relaxation, cause DNA damage, and consequently activate the DNA repair pathway. Additionally, anti-migration and anti-invasion abilities of Metachromin C were confirmed using the transwell assay. It also inhibited angiogenesis in human endothelial cells by reducing cell proliferation, migration, and disrupting tube formation. Moreover, Metachromin C dose-dependently inhibited the growth of intersegmental vessels, subintestinal vessels, and the caudal vein plexus in a zebrafish embryo model, confirming its inhibitory effect on new vessel formation in vivo. Taken together, Metachromin C could not only inhibit the growth of pancreatic cancer cells but also act as an anti-angiogenic compound simultaneously.

DNA structural changes (photo)induced by tricarbonyl (pterin)rhenium(I) complex

We report on interactions of different types of DNA molecules including double-stranded and plasmid DNA as well as polynucleotides (poly[dGdC]2 and poly[dAdT]2) with fac-[ReI(CO)3(pterin)(H2O)] (or Reptr) complex. The interaction was characterized spectroscopically and changes in the plasmid structure were verified by both electrophoresis and AFM microscopy. For comparative reasons, two others related tricarbonyl rhenium(I) complexes, fac-[(4,4′-bpy)ReI(CO)3(dppz)]+ (or Redppz) and fac-[(CF3SO3)ReI(CO)3(2,2′-bpy)] (or Rebpy) were also studied to further explore the influence of the different co-ligands on the interaction and DNA (photo)damage. Data reported herein suggests that DNA molecules can be structurally modified either by direct interaction with Re(I) complexes in their ground states inducing DNA relaxation, and/or through photoinduced cross-linking processes. The chemical nature of the co-ligands modulates the extent of the damage observed.

Synergy between human DNA ligase I and topoisomerase 1 unveils new therapeutic strategy for the management of colorectal cancer

DNA topoisomerase 1 (Topo 1) is a pivotal player in various DNA processes, including replication, repair, and transcription. It serves as a target for anticancer drugs like camptothecin and its derivatives (Topotecan and SN-38/Irinotecan). However, the emergence of drug resistance and the associated adverse effects, such as alopecia, anemia, dyspnea, fever, chills, and painful or difficult urination, pose significant challenges in Topo 1-targeted therapy, necessitating urgent attention. Human DNA Ligase 1 (hLig I), recognized primarily for its role in DNA replication and repair of DNA breaks, intriguingly exhibits a DNA relaxation activity akin to Topo 1. This raised the hypothesis that hLig I might compensate for Topo 1 inhibition, contributing to resistance against Topo 1 inhibitors. To explore this hypothesis, we assessed the efficacy of hLig I inhibition alone and in combination with Topo 1 in cancer cells. As anticipated, the overexpression of hLig I was observed after Topo 1 inhibition in colorectal cancer cells, affirming our hypothesis. Previously identified as an inhibitor of hLig I’s DNA relaxation activity, compound 27 (C 27), when combined with Topotecan, demonstrated a synergistic antiproliferative effect on colorectal cancer cells. Notably, cells with downregulated hLig I (via siRNA, inhibitors, or genetic manipulation) exhibited significantly heightened sensitivity to Topotecan. This observation strongly supports the concept that hLig I contribute to resistance against clinically relevant Topo 1 inhibitors in colorectal cancers. In conclusion, our findings offer evidence for the synergistic impact of combining hLig I inhibitors with Topotecan in the treatment of colorectal cancers, providing a promising strategy to overcome resistance to Topo 1 inhibitors. Communicated by Ramaswamy H. Sarma