Search Results (12)

Stabilization of a G-quadruplex (G4) in the promotor of the c-MYC proto-oncogene leads to inhibition of gene expression, and it thus represents a potentially attractive new strategy for cancer treatment. However, most G4 stabilizers show little selectivity among the many G4s present in the cellular complement of DNA and RNA. Intriguingly, a crescent-shaped cell-penetrating thiazole peptide, TH3, preferentially stabilizes the c-MYC G4 over other promotor G4s, but the mechanisms leading to this selective binding remain obscure. To investigate these mechanisms at the atomic level, we performed an in silico comparative investigation of the binding of TH3 and its analogue TH1 to the G4s from the promotors of c-MYC, c-KIT1, c-KIT2, and BCL2. Molecular docking and molecular dynamics simulations, combined with in-depth analyses of non-covalent interactions and bulk and per-nucleotide binding free energies, revealed that both TH3 and TH1 can induce the formation of a sandwich-like framework through stacking with both the top and bottom G-tetrads of the c-MYC G4 and the adjacent terminal capping nucleotides. This framework produces enhanced binding affinities for c-MYC G4 relative to other promotor G4s, with TH3 exhibiting an outstanding binding priority. Van der Waals interactions were identified to be the key factor in complex formation in all cases. Collectively, our findings fully agree with available experimental data. Therefore, the identified mechanisms leading to specific binding of TH3 towards c-MYC G4 provide valuable information to guide the development of new selective G4 stabilizers. Full article

Chronic exposure to ultraviolet (UV) radiation is known to induce the formation of DNA photo-adducts, including cyclobutane pyrimidine dimers (CPDs) and Dewar valence derivatives (DVs). While CPDs usually occur at higher frequency than DVs, recent studies have shown that the latter display superior selectivity and significant stability in interaction with the human DNA/topoisomerase 1 complex (TOP1). With the aim to deeply investigate the mechanism of interaction of DVs with TOP1, we report here four all-atom molecular dynamic simulations spanning one microsecond. These simulations are focused on the stability and conformational changes of two DNA/TOP1-DV complexes in solution, the data being compared with the biomimetic thymine dimer counterparts. Results from root-mean-square deviation (RMSD) and root-mean-square fluctuation (RMSF) analyses unequivocally confirmed increased stability of the DNA/TOP1-DV complexes throughout the simulation duration. Detailed interaction analyses, uncovering the presence of salt bridges, hydrogen bonds, water-mediated interactions, and hydrophobic interactions, as well as pinpointing the non-covalent interactions within the complexes, enabled the identification of specific TOP1 residues involved in the interactions over time and suggested a potential TOP1 inhibition mechanism in action. Full article

Etoposide (ETO) is an anticancer drug that targets topoisomerase II (TOP2). It stabilizes a normally transient TOP2–DNA covalent complex (TOP2cc), thus leading to DNA double-strand breaks (DSBs). Tyrosyl-DNA phosphodiesterases two (TDP2) is directly involved in the repair of TOP2cc by removing phosphotyrosyl peptides from 5′-termini of DSBs. Recent studies suggest that additional factors are required for TOP2cc repair, which include the proteasome and the zinc finger protein associated with TDP2 and TOP2, named ZATT. ZATT may alter the conformation of TOP2cc in a way that renders the accessibility of TDP2 for TOP2cc removal. In this study, our genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screens revealed that ZATT also has a TDP2-independent role in promoting cell survival following ETO treatment. ZATT KO cells showed relatively higher ETO sensitivity than TDP2-KO cells, and ZATT/TDP2 DKO cells displayed additive hypersensitivity to ETO treatment. The study using a series of deletion mutants of ZATT determined that the N-terminal 1–168 residues of ZATT are required for interaction with TOP2 and this interaction is critical to ETO sensitivity. Moreover, depletion of ZATT resulted in accelerated TOP2 degradation after ETO or cycloheximide (CHX) treatment, suggesting that ZATT may increase TOP2 stability and likely participate in TOP2 turnover. Taken together, this study suggests that ZATT is a critical determinant that dictates responses to ETO treatment and targeting. ZATT is a promising strategy to increase ETO efficacy for cancer therapy. Full article

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is an important repair enzyme that removes various covalent adducts from the 3′ end of DNA. Particularly, covalent complexes of topoisomerase 1 (TOP1) with DNA stabilized by DNA damage or by various chemical agents are an examples of such adducts. Anticancer drugs such as the TOP1 poisons topotecan and irinotecan are responsible for the stabilization of these complexes. TDP1 neutralizes the effect of these anticancer drugs, eliminating the DNA adducts. Therefore, the inhibition of TDP1 can sensitize tumor cells to the action of TOP1 poisons. This review contains information about methods for determining the TDP1 activity, as well as describing the inhibitors of these enzyme derivatives of natural biologically active substances, such as aminoglycosides, nucleosides, polyphenolic compounds, and terpenoids. Data on the efficiency of combined inhibition of TOP1 and TDP1 in vitro and in vivo are presented. Full article

Topoisomerase 1 (TOP1) is an enzyme that regulates DNA topology and is essential for replication, recombination, and other processes. The normal TOP1 catalytic cycle involves the formation of a short-lived covalent complex with the 3′ end of DNA (TOP1 cleavage complex, TOP1cc), which can be stabilized, resulting in cell death. This fact substantiates the effectiveness of anticancer drugs—TOP1 poisons, such as topotecan, that block the relegation of DNA and fix TOP1cc. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is able to eliminate TOP1cc. Thus, TDP1 interferes with the action of topotecan. Poly(ADP-ribose) polymerase 1 (PARP1) is a key regulator of many processes in the cell, such as maintaining the integrity of the genome, regulation of the cell cycle, cell death, and others. PARP1 also controls the repair of TOP1cc. We performed a transcriptomic analysis of wild type and PARP1 knockout HEK293A cells treated with topotecan and TDP1 inhibitor OL9-119 alone and in combination. The largest number of differentially expressed genes (DEGs, about 4000 both up- and down-regulated genes) was found in knockout cells. Topotecan and OL9-119 treatment elicited significantly fewer DEGs in WT cells and negligible DEGs in PARP1-KO cells. A significant part of the changes caused by PARP1-KO affected the synthesis and processing of proteins. Differences under the action of treatment with TOP1 or TDP1 inhibitors alone were found in the signaling pathways for the development of cancer, DNA repair, and the proteasome. The drug combination resulted in DEGs in the ribosome, proteasome, spliceosome, and oxidative phosphorylation pathways. Full article

The use of cancer chemotherapy sensitizers is a promising approach to induce the effect of clinically used anticancer treatments. One of the interesting targets is Tyrosyl-DNA Phosphodiesterase 1 (Tdp1), a DNA-repair enzyme, that may prevent the action of clinical Topoisomerase 1 (Top1) inhibitors, such as topotecan (Tpc). Tdp1 eliminates covalent Top1-DNA (Top1c) complexes that appear under the action of topotecan and determines the cytotoxic effect of this drug. We hypothesize that Tdp1 inhibition would sensitize cells towards the effect of Tpc. Herein, we report the synthesis and study of lipophilic derivatives of purine nucleosides that efficiently suppress Tdp1 activity, with IC₅₀ values in the 0.3–22.0 μM range. We also showed that this compound class can enhance DNA damage induced by topotecan in vitro by Comet assay on human cell lines HeLa and potentiate the antitumor effect of topotecan in vivo on a mice ascitic Krebs-2 carcinoma model. Thereby, this type of compound may be useful to develop drugs, that sensitize the effect of topotecan and reduce the required dose and, as a result, side effects. Full article

Inhibiting tyrosyl-DNA phosphodiesterase 1 (TDP1) is a promising strategy for increasing the effectiveness of existing antitumor therapy since it can remove the DNA lesions caused by anticancer drugs, which form covalent complexes with topoisomerase 1 (TOP1). Here, new adamantane–monoterpene conjugates with a 1,2,4-triazole or 1,3,4-thiadiazole linker core were synthesized, where (+)-and (−)-campholenic and (+)-camphor derivatives were used as monoterpene fragments. The campholenic derivatives 14a–14b and 15a–b showed activity against TDP1 at a low micromolar range with IC₅₀ ~5–6 μM, whereas camphor-containing compounds 16 and 17 were ineffective. Surprisingly, all the compounds synthesized demonstrated a clear synergy with topotecan, a TOP1 poison, regardless of their ability to inhibit TDP1. These findings imply that different pathways of enhancing topotecan toxicity other than the inhibition of TDP1 can be realized. Full article

Antimicrobial resistance is among the top global health problems with antibacterial resistance currently representing the major threat both in terms of occurrence and complexity. One reason current treatments of bacterial diseases are ineffective is the occurrence of protective and resistant biofilm structures. Phytochemicals are currently being reviewed for newer anti-virulence agents. In the present study, we aimed to investigate the anti-virulence activity of 3,3′-diindolylmethane (DIM), a bioactive cruciferous phytochemical. Using a series of in vitro assays on major Gram-negative pathogens, including transcriptomic analysis, and in vivo porcine wound studies as well as in silico experiments, we show that DIM has anti-biofilm activity. Following DIM treatment, our findings show that biofilm formation of two of the most prioritized bacterial pathogens Acinetobacter baumannii and Pseudomonas aeruginosa was inhibited respectively by 65% and 70%. Combining the antibiotic tobramycin with DIM enabled a high inhibition (94%) of P. aeruginosa biofilm. A DIM-based formulation, evaluated for its wound-healing efficacy on P. aeruginosa-infected wounds, showed a reduction in its bacterial bioburden, and wound size. RNA-seq was used to evaluate the molecular mechanism underlying the bacterial response to DIM. The gene expression profile encompassed shifts in virulence and biofilm-associated genes. A network regulation analysis showed the downregulation of 14 virulence-associated super-regulators. Quantitative real-time PCR verified and supported the transcriptomic results. Molecular docking and interaction profiling indicate that DIM can be accommodated in the autoinducer- or DNA-binding pockets of the virulence regulators making multiple non-covalent interactions with the key residues that are involved in ligand binding. DIM treatment prevented biofilm formation and destroyed existing biofilm without affecting microbial death rates. This study provides evidence for bacterial virulence attenuation by DIM. Full article

Usnic acid (UA) is a secondary metabolite of lichens that exhibits a wide range of biological activities. Previously, we found that UA derivatives are effective inhibitors of tyrosyl-DNA phosphodiesterase 1 (TDP1). It can remove covalent complex DNA-topoisomerase 1 (TOP1) stabilized by the TOP1 inhibitor topotecan, neutralizing the effect of the drugs. TDP1 removes damage at the 3′ end of DNA caused by other anticancer agents. Thus, TDP1 is a promising therapeutic target for the development of drug combinations with topotecan, as well as other drugs for cancer treatment. Ten new UA enamino derivatives with variation in the terpene fragment and substituent of the UA backbone were synthesized and tested as TDP1 inhibitors. Four compounds, 11a-d, had IC₅₀ values in the 0.23–0.40 μM range. Molecular modelling showed that 11a-d, with relatively short aliphatic chains, fit to the important binding domains. The intrinsic cytotoxicity of 11a-d was tested on two human cell lines. The compounds had low cytotoxicity with CC₅₀ ≥ 60 μM for both cell lines. 11a and 11c had high inhibition efficacy and low cytotoxicity, and they enhanced topotecan’s cytotoxicity in cancerous HeLa cells but reduced it in the non-cancerous HEK293A cells. This “protective” effect from topotecan on non-cancerous cells requires further investigation. Full article

DNA topoisomerase II (TOP2) activity involves a normally transient double-strand break intermediate in which the enzyme is coupled to DNA via a 5′-phosphotyrosyl bond. However, etoposide and other topoisomerase drugs poison the enzyme by stabilising this enzyme-bridged break, resulting in the accumulation of TOP2-DNA covalent complexes with cytotoxic consequences. The phosphotyrosyl diesterase TDP2 appears to be required for efficient repair of this unusual type of DNA damage and can remove 5′-tyrosine adducts from a double-stranded oligonucleotide substrate. Here, we adapt the trapped in agarose DNA immunostaining (TARDIS) assay to investigate the role of TDP2 in the removal of TOP2-DNA complexes in vitro and in cells. We report that TDP2 alone does not remove TOP2-DNA complexes from genomic DNA in vitro and that depletion of TDP2 in cells does not slow the removal of TOP2-DNA complexes. Thus, if TDP2 is involved in repairing TOP2 adducts, there must be one or more prior steps in which the protein-DNA complex is processed before TDP2 removes the remaining 5′ tyrosine DNA adducts. While this is partly achieved through the degradation of TOP2 adducts by the proteasome, a proteasome-independent mechanism has also been described involving the SUMOylation of TOP2 by the ZATT E3 SUMO ligase. The TARDIS assay was also adapted to measure the effect of TDP2 knockdown on levels of SUMOylated TOP2-DNA complexes, which together with levels of double strand breaks were unaffected in K562 cells following etoposide exposure and proteasomal inhibition. Full article

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is involved in DNA repair pathways as it mends the topoisomerase I—DNA covalent complexes. In plants, a small Tdp1 gene family, composed by Tdp1α and Tdp1β genes, was identified, but the roles of these genes in abiotic stress responses are not fully understood. To investigate their specific stress response patterns, the present study made use of bioinformatic and molecular tools to look into the Tdp1β gene function, so far described only in the plant kingdom, and compare it with Tdp1α gene coding for the canonical, highly conserved α isoform. The expression profiles of Tdp1α and Tdp1β genes were examined under abiotic stress conditions (cold, heat, high osmolarity, salt, and UV-B) in two model species, Arabidopsis thaliana and Medicago truncatula. The two isoforms of topoisomerase I (TOP1α and TOP1β) were also taken into consideration in view of their known roles in DNA metabolism and cell proliferation. Data relative to gene expression in Arabidopsis were retrieved from the AtGenExpress microarray dataset, while quantitative Real-Time PCR was carried out to evaluate the stress response in M. truncatula cell cultures. These analyses revealed that Tdp1β gene expression was enhanced during the first hour of treatment, whereas Tdp1α enhanced expression succeeded at subsequent timepoints. In agreement with the gene-specific responses to abiotic stress conditions, the promoter regions of Tdp1α and Tdp1β genes are well equipped with stress-related cis-elements. An in-depth bioinformatic characterization of the HIRAN motif, a distinctive feature of the Tdp1β protein, showed its wide distribution in chromatin remodeling and DNA repair proteins. The reported data suggests that Tdp1β functions in the early response to abiotic stresses. Full article

Evodiamine (EVO), an alkaloidal compound isolated from Evodia rutaecarpa (Juss.), has been reported to affect many physiological functions. Topoisomerase inhibitors have been developed in a variety of clinical applications. In the present study, we report the topoisomerase I (TopI) inhibitory activity of EVO, which may have properties that lead to improved therapeutic benefits. EVO is able to inhibit supercoiled plasmid DNA relaxation catalyzed by TopI. Upon treatment 0~10 μM EVO TopI was depleted in MCF-7 breast cancer cells in a concentration-dependent and time-dependent manner in 0~120 min. A K-SDS precipitation assay was performed to measure the extent of Top I-trapped chromosomal DNA. The ability of EVO to cause the formation of a TopI-DNA complex increased in a concentration-dependent manner, in that the DNA trapped increased by 24.2% in cells treated with 30 μM. The results suggest that EVO inhibits TopI by stabilizing the enzyme and DNA covalent complex. Full article