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MBio Apr 2024Type II topoisomerase utilizes the energy from ATP hydrolysis to alter DNA topology during genome replication and transcription. The ATPase domain of this enzyme is...
UNLABELLED
Type II topoisomerase utilizes the energy from ATP hydrolysis to alter DNA topology during genome replication and transcription. The ATPase domain of this enzyme is required for ATP hydrolysis and plays a crucial role in coupling DNA binding and ATP turnover with the DNA strand passage reaction. The African swine fever virus (ASFV) specifically encodes a topoisomerase II (topo II), which is critical for viral replication and an attractive target for antiviral development. Here, we present a high-resolution crystal structure of the ASFV topo II ATPase domain complexed with the substrate analog AMPPNP. Structural comparison reveals that the ASFV topo II ATPase domain shares a conserved overall structure with its homologs from eukaryotes and prokaryotes but also has three characteristic regions, including the intra-molecular interface formed by the ATP-lid and QTK loop as well as helix α9, the K-loop in the transducer domain, and the antennae-like α-helix at the ATP binding domain. Mutating the key residues within these three regions impairs or abolishes the basal and DNA-stimulated ATPase activities and reduces or eliminates the relaxation activity of the holoenzyme. Our data indicate that all three regions are functionally important for the ATPase and relaxation activities and strongly suggest that ATP hydrolysis, DNA binding, and strand passage are highly coupled and managed by the allosteric coordination of multiple domains of the type II topoisomerase. Moreover, we find a promising druggable pocket in the dimeric interface of the ASFV topo II ATPase domain, which will benefit future anti-ASFV drug development.
IMPORTANCE
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.
Topics: Swine; Animals; African Swine Fever Virus; Adenylyl Imidodiphosphate; DNA Topoisomerases, Type II; DNA; Adenosine Triphosphatases
PubMed: 38411066
DOI: 10.1128/mbio.03086-23 -
Frontiers in Chemistry 2024The main objective of this study is to synthesize and characterize of a new three complexes of Pd (II), Cu (II), and Cu (I) metal ions with novel ligand...
Synthesis, characterization, PXRD studies, and theoretical calculation of the effect of gamma irradiation and antimicrobial studies on novel Pd(II), Cu(II), and Cu(I) complexes.
The main objective of this study is to synthesize and characterize of a new three complexes of Pd (II), Cu (II), and Cu (I) metal ions with novel ligand ((Z)-2-(phenylamino)-N'-(thiophen-2-ylmethylene)acetohydrazide) HL. The structural composition of new compounds was assessed using several analytical techniques including FT-IR, H-NMR, electronic spectra, powder X-ray diffraction, and thermal behavior analysis. The Gaussian09 program employed the Density Functional Theory (DFT) approach to optimize the geometry of all synthesized compounds, therefore obtaining the most favorable structures and crucial parameters. An investigation was conducted to examine the impact of γ-irradiation on ligands and complexes. Before and after γ-irradiation, the antimicrobial efficiency was investigated for the activity of ligands and their chelates. The Cu(I) complex demonstrated enhanced antibacterial activity after irradiation, as well as other standard medications such as ampicillin and gentamicin. Similarly, the Cu(I) complex exhibited superior activity against antifungal species relative to the standard drug Nystatin. The docking investigation utilized the target location of the topoisomerase enzyme (2xct) chain A.
PubMed: 38410818
DOI: 10.3389/fchem.2024.1357330 -
International Journal of Molecular... Feb 2024Fluoroquinolones are potentially active against . Rapidly increased minimum inhibitory concentrations (MICs) and emerging point mutations in the quinolone...
Fluoroquinolones are potentially active against . Rapidly increased minimum inhibitory concentrations (MICs) and emerging point mutations in the quinolone resistance-determining regions (QRDRs) following exposure to fluoroquinolones have been reported in . We aimed to investigate point mutations in QRDRs through exposure to levofloxacin (1 × MIC) combinations with different concentrations (0.5× and 1 × MIC) of minocycline, rifampin, cefoperazone/sulbactam, or sulfamethoxazole/trimethoprim in comparison with exposure to levofloxacin alone. Of the four isolates that were clinically collected, lower MICs of levofloxacin were disclosed in cycle 2 and 3 of induction and selection in all levofloxacin combination groups other than levofloxacin alone (all = 0.04). Overall, no mutations were discovered in and throughout the multicycles inducted by levofloxacin and all its combinations. Regarding the vastly increased MICs, the second point mutations in and/or in one isolate (strain no. 1) occurred in cycle 2 following exposure to levofloxacin plus 0.5 × MIC minocycline, but they were delayed appearing in cycle 5 following exposure to levofloxacin plus 1 × MIC minocycline. Similarly, the second point mutation in and/or occurred in another isolate (strain no. 3) in cycle 4 following exposure to levofloxacin plus 0.5 × MIC sulfamethoxazole/trimethoprim, but no mutation following exposure to levofloxacin plus 1 × MIC sulfamethoxazole/trimethoprim was disclosed. In conclusion, the rapid selection of mutants with high MICs after levofloxacin exposure could be effectively delayed or postponed by antimicrobial combination with other in vitro active antibiotics.
Topics: Levofloxacin; Minocycline; DNA Gyrase; Anti-Bacterial Agents; Fluoroquinolones; Microbial Sensitivity Tests; Mutation; Sulfamethoxazole; Trimethoprim; Drug Resistance, Bacterial; Flavobacteriaceae
PubMed: 38396892
DOI: 10.3390/ijms25042215 -
International Journal of Molecular... Feb 2024The in-silico strategy of identifying novel uses for already existing drugs, known as drug repositioning, has enhanced drug discovery. Previous studies have shown a...
The in-silico strategy of identifying novel uses for already existing drugs, known as drug repositioning, has enhanced drug discovery. Previous studies have shown a positive correlation between expression changes induced by the anticancer agent trabectedin and those caused by irinotecan, a topoisomerase I inhibitor. Leveraging the availability of transcriptional datasets, we developed a general in-silico drug-repositioning approach that we applied to investigate novel trabectedin synergisms. We set a workflow allowing the identification of genes selectively modulated by a drug and possible novel drug interactions. To show its effectiveness, we selected trabectedin as a case-study drug. We retrieved eight transcriptional cancer datasets including controls and samples treated with trabectedin or its analog lurbinectedin. We compared gene signature associated with each dataset to the 476,251 signatures from the Connectivity Map database. The most significant connections referred to mitomycin-c, topoisomerase II inhibitors, a PKC inhibitor, a Chk1 inhibitor, an antifungal agent, and an antagonist of the glutamate receptor. Genes coherently modulated by the drugs were involved in cell cycle, PPARalpha, and Rho GTPases pathways. Our in-silico approach for drug synergism identification showed that trabectedin modulates specific pathways that are shared with other drugs, suggesting possible synergisms.
Topics: Trabectedin; Tetrahydroisoquinolines; Dioxoles; Antineoplastic Agents; Drug Synergism
PubMed: 38396735
DOI: 10.3390/ijms25042059 -
BMC Complementary Medicine and Therapies Feb 2024DNA topoisomerases regulate conformational changes in DNA topology during normal cell growth, such as replication, transcription, recombination, and repair, and may be...
DNA topoisomerases regulate conformational changes in DNA topology during normal cell growth, such as replication, transcription, recombination, and repair, and may be targeted for anticancer drugs. A DNA topology assay was used to investigate DNA-damaging/protective activities of extracts from Habanero Red (HR), Habanero Maya Red (HMR), Trinidad Moruga Scorpion (TMS), Jalapeno (J), Serrano pepper (SP), Habanero Red Savina (HRS), Bhut Jolokia (BJ), and Jamaica Rosso (JR) peppers, demonstrating their inhibitory effect on the relaxation of pBR by Topo I. DNA topoisomerase II (Topo II) is proven therapeutic target of anticancer drugs. Complete inhibition of Topo II was observed for samples TMS, HR, and HMR. Extracts J and SP had the lowest capsaicin and dihydrocapsaicin content compared to other peppers. HR, HMR, TMS, J, S, HRS, BJ, JR extracts showed the anticancer effect, examined by MTS and xCell assay on the in vitro culture of human colon carcinoma cell line HCT116.
Topics: Humans; Capsaicin; Capsicum; Antineoplastic Agents; DNA
PubMed: 38383414
DOI: 10.1186/s12906-024-04394-5 -
Cell Reports Feb 2024We trapped catalytically engaged topoisomerase IIβ (TOP2B) in covalent DNA cleavage complexes (TOP2Bccs) and mapped their positions genome-wide in cultured mouse...
We trapped catalytically engaged topoisomerase IIβ (TOP2B) in covalent DNA cleavage complexes (TOP2Bccs) and mapped their positions genome-wide in cultured mouse cortical neurons. We report that TOP2Bcc distribution varies with both nucleosome and compartmental chromosome organization. While TOP2Bccs in gene bodies correlate with their level of transcription, highly expressed genes that lack the usually associated chromatin marks, such as H3K36me3, show reduced TOP2Bccs, suggesting that histone posttranslational modifications regulate TOP2B activity. Promoters with high RNA polymerase II occupancy show elevated TOP2B chromatin immunoprecipitation sequencing signals but low TOP2Bccs, indicating that TOP2B catalytic engagement is curtailed at active promoters. Surprisingly, either poisoning or inhibiting TOP2B increases nascent transcription at most genes and enhancers but reduces transcription within long genes. These effects are independent of transcript length and instead correlate with the presence of intragenic enhancers. Together, these results clarify how cells modulate the catalytic engagement of topoisomerases to affect transcription.
Topics: Animals; Mice; Catalysis; Chromatin; Chromatin Immunoprecipitation Sequencing; DNA Cleavage; Neurons
PubMed: 38377005
DOI: 10.1016/j.celrep.2024.113809 -
Scientific Reports Feb 2024Fungal pathogens are considered as serious factors for deadly diseases and are a case of medical concern. Invasive fungal infections also complicate the clinical course...
Fungal pathogens are considered as serious factors for deadly diseases and are a case of medical concern. Invasive fungal infections also complicate the clinical course of COVID-19, leading to a significant increase in mortality. Furthermore, fungal strains' multidrug resistance has increased the demand for antifungals with a different mechanism of action. The present study aimed to identify antifungal compounds targeting yeast topoisomerase II (yTOPOII) derived from well-known human topoisomerase II (hTOPOII) poisons C-1305 and C-1311. Two sets of derivatives: triazoloacridinones (IKE1-8) and imidazoacridinones (IKE9-14) were synthetized and evaluated with a specific emphasis on the molecular mechanism of action. Our results indicated that their effectiveness as enzyme inhibitors was not solely due to intercalation ability but also as a result of influence on catalytic activity by the formation of covalent complexes between plasmid DNA and yTOPOII. Lysine conjunction increased the strength of the compound's interaction with DNA and improved penetration into the fungal cells. Triazoloacridinone derivatives in contrast to starting compound C-1305 exhibited moderate antifungal activity and at least twice lower cytotoxicity. Importantly, compounds (IKE5-8) were not substrates for multidrug ABC transporters whereas a derivative conjugated with lysine (IKE7), showed the ability to overcome C. glabrata fluconazole-resistance (MIC 32-64 µg mL).
Topics: Humans; Antifungal Agents; Lysine; Fluconazole; ATP-Binding Cassette Transporters; Candida glabrata; DNA; Microbial Sensitivity Tests
PubMed: 38351313
DOI: 10.1038/s41598-024-54252-0 -
Nucleic Acids Research May 2024In cancer therapy, DNA intercalators are mainly known for their capacity to kill cells by inducing DNA damage. Recently, several DNA intercalators have attracted much...
In cancer therapy, DNA intercalators are mainly known for their capacity to kill cells by inducing DNA damage. Recently, several DNA intercalators have attracted much interest given their ability to inhibit RNA Polymerase I transcription (BMH-21), evict histones (Aclarubicin) or induce chromatin trapping of FACT (Curaxin CBL0137). Interestingly, these DNA intercalators lack the capacity to induce DNA damage while still retaining cytotoxic effects and stabilize p53. Herein, we report that these DNA intercalators impact chromatin biology by interfering with the chromatin stability of RNA polymerases I, II and III. These three compounds have the capacity to induce degradation of RNA polymerase II and they simultaneously enable the trapping of Topoisomerases TOP2A and TOP2B on the chromatin. In addition, BMH-21 also acts as a catalytic inhibitor of Topoisomerase II, resembling Aclarubicin. Moreover, BMH-21 induces chromatin trapping of the histone chaperone FACT and propels accumulation of Z-DNA and histone eviction, similarly to Aclarubicin and CBL0137. These DNA intercalators have a cumulative impact on general transcription machinery by inducing accumulation of topological defects and impacting nuclear chromatin. Therefore, their cytotoxic capabilities may be the result of compounding deleterious effects on chromatin homeostasis.
Topics: Humans; Antigens, Neoplasm; Carbazoles; Chromatin; Diketopiperazines; DNA; DNA Damage; DNA Topoisomerases, Type II; DNA-Binding Proteins; High Mobility Group Proteins; Histones; Intercalating Agents; Poly-ADP-Ribose Binding Proteins; RNA Polymerase I; RNA Polymerase II; RNA Polymerase III; Topoisomerase II Inhibitors; Transcription, Genetic; Transcriptional Elongation Factors; Aclarubicin
PubMed: 38340348
DOI: 10.1093/nar/gkae069 -
BioRxiv : the Preprint Server For... Jan 2024Human type-II topoisomerases, TOP2A and TOP2B, remove transcription associated DNA supercoiling, thereby affecting gene-expression programs, and have recently been...
Human type-II topoisomerases, TOP2A and TOP2B, remove transcription associated DNA supercoiling, thereby affecting gene-expression programs, and have recently been associated with 3D genome architecture. Here, we study the regulatory roles of TOP2 paralogs in response to estrogen, which triggers an acute transcriptional induction that involves rewiring of genome organization. We find that, whereas TOP2A facilitates transcription, as expected for a topoisomerase, TOP2B limits the estrogen response. Consistent with this, TOP2B activity is locally downregulated upon estrogen treatment to favor the establishment and stabilization of regulatory chromatin contacts, likely through an accumulation of DNA supercoiling. We show that estrogen-mediated inhibition of TOP2B requires estrogen receptor α (ERα), a non-catalytic function of TOP2A, and the action of the atypical SUMO-ligase ZATT. This mechanism of topological transcriptional-control, which may be shared by additional gene-expression circuits, highlights the relevance of DNA topoisomerases as central actors of genome dynamics.
PubMed: 38328138
DOI: 10.1101/2024.01.22.576640 -
Scientific Reports Feb 2024The spread of fluoroquinolone (FQ) resistance in Acinetobacter baumannii represents a critical health threat. This study aims to overcome FQ resistance in A. baumannii...
The spread of fluoroquinolone (FQ) resistance in Acinetobacter baumannii represents a critical health threat. This study aims to overcome FQ resistance in A. baumannii via the formulation of polymeric nanoFQs. Herein, 80 A. baumannii isolates were obtained from diverse clinical sources. All A. baumannii isolates showed high resistance to most of the investigated antimicrobials, including ciprofloxacin (CIP) and levofloxacin (LEV) (97.5%). FQ resistance-determining regions of the gyrA and parC genes were the most predominant resistant mechanism, harbored by 69 (86.3%) and 75 (93.8%) of the isolates, respectively. Additionally, plasmid-mediated quinolone resistance genes aac(6')-Ib and qnrS were detected in 61 (76.3%) and 2 (2.5%) of the 80 isolates, respectively. The CIP- and LEV-loaded poly ε-caprolactone (PCL) nanoparticles, F and F, respectively, showed a 1.5-6- and 6-12-fold decrease in the MIC, respectively, against the tested isolates. Interestingly, the time kill assay demonstrated that MICs of F and F completely killed A. baumannii isolates after 5-6 h of treatment. Furthermore, F and F were found to be efficient in overcoming the FQ resistance mediated by the efflux pumps in A. baumannii isolates as revealed by decreasing the MIC four-fold lower than that of free CIP and LEV, respectively. Moreover, F and F at 1/2 and 1/4 MIC significantly decreased biofilm formation by 47-93% and 69-91%, respectively. These findings suggest that polymeric nanoparticles can restore the effectiveness of FQs and represent a paradigm shift in the fight against A. baumannii isolates.
Topics: Ciprofloxacin; Fluoroquinolones; Levofloxacin; Acinetobacter baumannii; Anti-Bacterial Agents; Microbial Sensitivity Tests; Biofilms; Drug Resistance, Bacterial; DNA Gyrase
PubMed: 38326515
DOI: 10.1038/s41598-024-53441-1