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Biochemistry Jun 2024DNA gyrases catalyze negative supercoiling of DNA, are essential for bacterial DNA replication, transcription, and recombination, and are important antibacterial targets...
DNA gyrases catalyze negative supercoiling of DNA, are essential for bacterial DNA replication, transcription, and recombination, and are important antibacterial targets in multiple pathogens, including , which in 2021 caused >1.5 million deaths worldwide. DNA gyrase is a tetrameric (AB) protein formed from two subunit types: gyrase A (GyrA) carries the breakage-reunion active site, whereas gyrase B (GyrB) catalyzes ATP hydrolysis required for energy transduction and DNA translocation. The GyrB ATPase domains dimerize in the presence of ATP to trap the translocated DNA (T-DNA) segment as a first step in strand passage, for which hydrolysis of one of the two ATPs and release of the resulting inorganic phosphate is rate-limiting. Here, dynamical-nonequilibrium molecular dynamics (D-NEMD) simulations of the dimeric 43 kDa N-terminal fragment of GyrB show how events at the ATPase site (dissociation/hydrolysis of bound nucleotides) are propagated through communication pathways to other functionally important regions of the GyrB ATPase domain. Specifically, our simulations identify two distinct pathways that respectively connect the GyrB ATPase site to the corynebacteria-specific C-loop, thought to interact with GyrA prior to DNA capture, and to the C-terminus of the GyrB transduction domain, which in turn contacts the C-terminal GyrB topoisomerase-primase (TOPRIM) domain responsible for interactions with GyrA and the centrally bound G-segment DNA. The connection between the ATPase site and the C-loop of dimeric GyrB is consistent with the unusual properties of DNA gyrase relative to those from other bacterial species.
Topics: Mycobacterium tuberculosis; DNA Gyrase; Molecular Dynamics Simulation; Adenosine Triphosphatases; Protein Domains; Adenosine Triphosphate; Bacterial Proteins; Signal Transduction
PubMed: 38742407
DOI: 10.1021/acs.biochem.4c00161 -
Phytomedicine : International Journal... Jul 2024Temozolomide (TMZ) resistance is the main obstacle faced by glioblastoma multiforme (GBM) treatment. Muscone, one of the primary active pharmacological ingredients of...
BACKGROUND
Temozolomide (TMZ) resistance is the main obstacle faced by glioblastoma multiforme (GBM) treatment. Muscone, one of the primary active pharmacological ingredients of Shexiang (Moschus), can cross the blood-brain barrier (BBB) and is being investigated as an antineoplastic medication. However, muscone treatment for GBM has received little research, and its possible mechanisms are still unclear.
PURPOSE
This study aims to evaluate the effect and the potential molecular mechanism of muscone on TMZ-resistant GBM cells.
METHODS
The differentially expressed genes (DEGs) between TMZ-resistant GBM cells and TMZ-sensitive GBM cells were screened using GEO2R. By progressively raising the TMZ concentration, a relatively stable TMZ-resistant human GBM cell line was established. The drug-resistance traits of U251-TR cells were assessed via the CCK-8 assay and Western Blot analysis of MGMT and TOP2A expression. Cell viability, cell proliferation, cell migration ability, and drug synergism were detected by the CCK-8 assay, colony formation assay, wound healing assay, and drug interaction relationship test, respectively. Anoikis was quantified by Calcein-AM/EthD-1 staining, MTT assay, and flow cytometry. Measurements of cell cycle arrest, apoptosis, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) were performed using cell cycle staining, Annexin V-FITC/PI labeling, JC-1 assay, and ROS assay, respectively. DNA damage was measured by TUNEL assay, alkaline comet assay, and γ-H2AX foci assay. GEPIA was used to investigate the link between the anoikis marker (FAK)/drug resistance gene and critical proteins in the EGFR/Integrin β1 signaling pathway. Molecular docking was used to anticipate the probable targets of muscone. The intracellular co-localization and expression of EGFR and FAK were shown using immunofluorescence. The U251-TR cell line stably overexpressing EGFR was constructed using lentiviral transduction to assess the involvement of EGFR-related signaling in anoikis resistance. Western Blot was employed to detect the expression of migration-related proteins, cyclins, anoikis-related proteins, DNA damage/repair-related proteins, and associated pathway proteins.
RESULTS
DEGs analysis identified 97 deregulated chemotherapy-resistant genes and 3779 upregulated genes in TMZ-resistant GBM cells. Subsequent experiments verified TMZ resistance and the hyper-expression of DNA repair-related genes (TOP2A and MGMT) in continuously low-dose TMZ-induced U251-TR cells. Muscone exhibited dose-dependent inhibition of U251-TR cell migration and proliferation, and its co-administration with TMZ showed the potential for enhanced therapeutic efficacy. By downregulating FAK, muscone reduced anoikis resistance in anchorage-independent U251-TR cells. It also caused cell cycle arrest in the G2/M phase by upregulating p21 and downregulating CDK1, CDK2, and Cyclin E1. Muscone-induced anoikis was accompanied by mitochondrial membrane potential collapse, ROS production, an increase in the BAX/Bcl-2 ratio, as well as elevated levels of Cytochrome c (Cyt c), cleaved caspase-9, and cleaved caspase-3. These findings indicated that muscone might trigger mitochondrial-dependent anoikis via ROS generation. Moreover, significant DNA damage, DNA double-strand breaks (DSBs), the formation of γ-H2AX foci, and a reduction in TOP2A expression are also associated with muscone-induced anoikis. Overexpression of EGFR in U251-TR cells boosted the expression of Integrin β1, FAK, β-Catenin, and TOP2A, whereas muscone suppressed the expression levels of EGFR, Integrin β1, β-Catenin, FAK, and TOP2A. Muscone may influence the expression of the key DNA repair enzyme, TOP2A, by suppressing the EGFR/Integrin β1/FAK pathway.
CONCLUSION
We first demonstrated that muscone suppressed TOP2A expression through the EGFR/Integrin β1/FAK pathway, hence restoring anoikis sensitivity in TMZ-resistant GBM cells. These data suggest that muscone may be a promising co-therapeutic agent for enhancing GBM treatment, particularly in cases of TMZ-resistant GBM with elevated TOP2A expression.
Topics: Humans; Glioblastoma; Temozolomide; Drug Resistance, Neoplasm; Signal Transduction; Cell Line, Tumor; Focal Adhesion Kinase 1; Anoikis; Integrin beta1; ErbB Receptors; DNA Topoisomerases, Type II; Cell Movement; Cell Proliferation; Poly-ADP-Ribose Binding Proteins; Cell Survival; Apoptosis; Reactive Oxygen Species
PubMed: 38723526
DOI: 10.1016/j.phymed.2024.155714 -
MSphere May 2024Cervimycins A-D are bis-glycosylated polyketide antibiotics produced by HKI 0179 with bactericidal activity against Gram-positive bacteria. In this study, cervimycin C...
Cervimycins A-D are bis-glycosylated polyketide antibiotics produced by HKI 0179 with bactericidal activity against Gram-positive bacteria. In this study, cervimycin C (CmC) treatment caused a spaghetti-like phenotype in 168, with elongated curved cells, which stayed joined after cell division, and exhibited a chromosome segregation defect, resulting in ghost cells without DNA. Electron microscopy of CmC-treated (3 × MIC) revealed swollen cells, misshapen septa, cell wall thickening, and a rough cell wall surface. Incorporation tests in indicated an effect on DNA biosynthesis at high cervimycin concentrations. Indeed, artificial downregulation of the DNA gyrase subunit B gene () increased the activity of cervimycin in agar diffusion tests, and, in high concentrations (starting at 62.5 × MIC), the antibiotic inhibited DNA gyrase supercoiling activity . To obtain a more global view on the mode of action of CmC, transcriptomics and proteomics of cervimycin treated versus untreated cells were performed. Interestingly, 3 × MIC of cervimycin did not induce characteristic responses, which would indicate disturbance of the DNA gyrase activity . Instead, cervimycin induced the expression of the CtsR/HrcA heat shock operon and the expression of autolysins, exhibiting similarity to the ribosome-targeting antibiotic gentamicin. In summary, we identified the DNA gyrase as a target, but at low concentrations, electron microscopy and omics data revealed a more complex mode of action of cervimycin, which comprised induction of the heat shock response, indicating protein stress in the cell.IMPORTANCEAntibiotic resistance of Gram-positive bacteria is an emerging problem in modern medicine, and new antibiotics with novel modes of action are urgently needed. Secondary metabolites from species are an important source of antibiotics, like the cervimycin complex produced by HKI 0179. The phenotypic response of and toward cervimycin C indicated a chromosome segregation and septum formation defect. This effect was at first attributed to an interaction between cervimycin C and the DNA gyrase. However, omics data of cervimycin treated versus untreated cells indicated a different mode of action, because the stress response did not include the SOS response but resembled the response toward antibiotics that induce mistranslation or premature chain termination and cause protein stress. In summary, these results point toward a possibly novel mechanism that generates protein stress in the cells and subsequently leads to defects in cell and chromosome segregation.
Topics: Anti-Bacterial Agents; Staphylococcus aureus; Microbial Sensitivity Tests; Streptomyces; Bacillus subtilis; Polyketides; Glycosides; Cell Wall; Proteomics; Bacterial Proteins; DNA Gyrase
PubMed: 38722162
DOI: 10.1128/msphere.00764-23 -
Biophysical Journal Jun 2024Type II topoisomerases (TopoIIs) are ubiquitous enzymes that are involved in crucial nuclear processes such as genome organization, chromosome segregation, and other DNA...
Type II topoisomerases (TopoIIs) are ubiquitous enzymes that are involved in crucial nuclear processes such as genome organization, chromosome segregation, and other DNA metabolic processes. These enzymes function as large, homodimeric complexes that undergo a complex cycle of binding and hydrolysis of two ATP molecules in their ATPase domains, which regulates the capture and passage of one DNA double-helix through a second, cleaved DNA molecule. This process requires the transmission of information about the state of the bound nucleotide over vast ranges in the TopoII complex. How this information is transmitted at the molecular level to regulate TopoII functions and how protein substitutions disrupt these mechanisms remains largely unknown. Here, we employed extensive microsecond-scale molecular dynamics simulations of the yeast TopoII enzyme in multiple nucleotide-bound states and with amino acid substitutions near both the N and C termini of the complex. Simulation results indicate that the ATPase domains are remarkably flexible on the sub-microsecond timescale and that these dynamics are modulated by the identity of the bound nucleotides and both local and distant amino acid substitutions. Network analyses point toward specific allosteric networks that transmit information about the hydrolysis cycle throughout the complex, which include residues in both the protein and the bound DNA molecule. Amino acid substitutions weaken many of these pathways. Together, our results provide molecular level details on how the TopoII catalytic cycle is controlled through nucleotide binding and hydrolysis and how mutations may disrupt this process.
Topics: Allosteric Regulation; DNA Topoisomerases, Type II; Molecular Dynamics Simulation; Saccharomyces cerevisiae; Protein Domains; Models, Molecular
PubMed: 38720465
DOI: 10.1016/j.bpj.2024.05.004 -
Molecular Pharmacology Jun 2024DNA topoisomerase II (TOP2; 170 kDa, TOP2/170) is an essential enzyme for proper chromosome dysjunction by producing transient DNA double-stranded breaks and is an...
DNA topoisomerase II (TOP2; 170 kDa, TOP2/170) is an essential enzyme for proper chromosome dysjunction by producing transient DNA double-stranded breaks and is an important target for DNA damage-stabilizing anticancer agents, such as etoposide. Therapeutic effects of TOP2 poisons can be limited due to acquired drug resistance. We previously demonstrated decreased TOP2/170 levels in an etoposide-resistant human leukemia K562 subline, designated K/VP.5, accompanied by increased expression of a C-terminal truncated TOP2 isoform (90 kDa; TOP2/90), which heterodimerized with TOP2/170 and was a determinant of resistance by exhibiting dominant-negative effects against etoposide activity. Based on 3'-rapid amplification of cDNA ends, we confirmed TOP2/90 as the translation product of a TOP2 mRNA in which a cryptic polyadenylation site (PAS) harbored in intron 19 (I19) was used. In this report, we investigated whether the resultant intronic polyadenylation (IPA) would be attenuated by blocking or mutating the I19 PAS, thereby circumventing acquired drug resistance. An antisense morpholino oligonucleotide was used to hybridize/block the PAS in TOP2 pre-mRNA in K/VP.5 cells, resulting in decreased TOP2/90 mRNA/protein levels in K/VP.5 cells and partially circumventing drug resistance. Subsequently, CRISPR/CRISPR-associated protein 9 with homology-directed repair was used to mutate the cryptic I19 PAS (AAA→AAA) to prevent IPA. Gene-edited clones exhibited increased TOP2/170 and decreased TOP2/90 mRNA/protein and demonstrated restored sensitivity to etoposide and other TOP2-targeted drugs. Together, results indicated that blocking/mutating a cryptic I19 PAS in K/VP.5 cells reduced IPA and restored sensitivity to TOP2-targeting drugs. SIGNIFICANCE STATEMENT: The results presented in this study indicate that CRISPR/CRISPR-associated protein 9 gene editing of a cryptic polyadenylation site (PAS) within I19 of the TOP2α gene results in the reversal of acquired resistance to etoposide and other TOP2-targeted drugs. An antisense morpholino oligonucleotide targeting the PAS also partially circumvented resistance.
Topics: Humans; Etoposide; DNA Topoisomerases, Type II; Drug Resistance, Neoplasm; K562 Cells; Polyadenylation; Introns; CRISPR-Cas Systems
PubMed: 38719474
DOI: 10.1124/molpharm.124.000868 -
International Journal of Biological... Jun 2024Type IIA DNA topoisomerases are molecular nanomachines responsible for controlling topological states of DNA molecules. Here, we explore the dynamic landscape of yeast...
Type IIA DNA topoisomerases are molecular nanomachines responsible for controlling topological states of DNA molecules. Here, we explore the dynamic landscape of yeast topoisomerase IIA during key stages of its catalytic cycle, focusing in particular on the events preceding the passage of the T-segment. To this end, we generated six configurations of fully catalytic yeast topo IIA, strategically inserted a T-segment into the N-gate in relevant configurations, and performed all-atom simulations. The essential motion of topo IIA protein dimer was characterized by rotational gyrating-like movement together with sliding motion within the DNA-gate. Both appear to be inherent properties of the enzyme and an inbuilt feature that allows passage of the T-segment through the cleaved G-segment. Coupled dynamics of the N-gate and DNA-gate residues may be particularly important for controlled and smooth passage of the T-segment and consequently the prevention of DNA double-strand breaks. QTK loop residue Lys367, which interacts with ATP and ADP molecules, is involved in regulating the size and stability of the N-gate. The unveiled features of the simulated configurations provide insights into the catalytic cycle of type IIA topoisomerases and elucidate the molecular choreography governing their ability to modulate the topological states of DNA topology.
Topics: DNA Topoisomerases, Type II; Molecular Dynamics Simulation; DNA; Saccharomyces cerevisiae; Protein Multimerization; Nucleic Acid Conformation
PubMed: 38714283
DOI: 10.1016/j.ijbiomac.2024.131991 -
Future Medicinal Chemistry 2024This study focused on designing and synthesizing novel derivatives of 3,5,8-trisubstituted coumarin. The synthesized compounds, particularly compound , exhibited...
This study focused on designing and synthesizing novel derivatives of 3,5,8-trisubstituted coumarin. The synthesized compounds, particularly compound , exhibited significant cytotoxic effects on MCF-7 cells, surpassing staurosporine, and reduced toxicity toward MCF-10A cells, highlighting potential pharmacological advantages. Further, compound altered the cell cycle and significantly increased apoptosis in MCF-7 cells, involving both early (41.7-fold) and late stages (33-fold), while moderately affecting necrotic signaling. The antitumor activity was linked to a notable reduction (4.78-fold) in topoisomerase IIβ expression. Molecular modeling indicated compound 's strong affinity for EGFR, human EGF2 and topoisomerase II proteins. These findings highlight compound as a multifaceted antitumor agent for breast cancer.
Topics: Humans; Coumarins; Breast Neoplasms; Antineoplastic Agents; Cell Proliferation; Female; Apoptosis; Drug Screening Assays, Antitumor; DNA Topoisomerases, Type II; Structure-Activity Relationship; MCF-7 Cells; Molecular Structure; Cell Line, Tumor; Cell Cycle; ErbB Receptors
PubMed: 38708686
DOI: 10.4155/fmc-2023-0375 -
Journal of Oleo Science 2024Launaea sarmentosa, also known as Sa Sam Nam, is a widely used remedy in Vietnamese traditional medicine and cuisine. However, the chemical composition and bioactivity...
Launaea sarmentosa, also known as Sa Sam Nam, is a widely used remedy in Vietnamese traditional medicine and cuisine. However, the chemical composition and bioactivity of its essential oil have not been elucidated yet. In this study, we identified 40 compounds (98.6% of total peak area) in the essential oil via GC-MS analysis at the first time. Among them, five main compounds including Thymohydroquinone dimethyl ether (52.4%), (E)-α-Atlantone (9.0%), Neryl isovalerate (6.6%), Davanol D2 (isomer 2) (3.9%), and trans-Sesquisabinene hydrate (3.9%) have accounted for 75.8% of total peak area. The anti-bacterial activity of the essential oil against 4 microorganisms including Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa has also investigated via agar well diffusion assay. The results showed that the essential oil exhibited a strong antibacterial activity against Bacillus subtilis with the inhibition zones ranging from 8.2 to 18.7 mm. To elucidate the anti-bacterial effect mechanism of the essential oil, docking study of five main compounds of the essential oil (Thymohydroquinone dimethyl ether, (E)-α-Atlantone, Neryl isovalerate, Davanol D2 (isomer 2), and trans-Sesquisabinene hydrate) against some key proteins for bacterial growth such as DNA gyrase B, penicillin binding protein 2A, tyrosyl-tRNA synthetase, and dihydrofolate reductase were performed. The results showed that the main constituents of essential oil were highly bound with penicillin binding protein 2A with the free energies ranging -27.7 to -44.8 kcal/mol, which suggests the relationship between the antibacterial effect of essential oil and the affinity of main compounds with penicillin binding protein. In addition, the free energies of main compounds of the essential oil with human cyclooxygenase 1, cyclooxygenase 2, and phospholipase A2, the crucial proteins related with inflammatory response were less than diclofenac, a non-steroidal antiinflammatory drug. These findings propose the essential oil as a novel and promising anti-bacterial and anti-inflammatory medicine or cosmetic products.
Topics: Molecular Docking Simulation; Anti-Bacterial Agents; Oils, Volatile; Bacillus subtilis; Staphylococcus aureus; Pseudomonas aeruginosa; Escherichia coli; Tetrahydrofolate Dehydrogenase; DNA Gyrase; Sesquiterpenes; Microbial Sensitivity Tests; Gas Chromatography-Mass Spectrometry; Pentanoic Acids; Hemiterpenes
PubMed: 38692900
DOI: 10.5650/jos.ess23254 -
Microbiology Spectrum Jun 2024This study aims to elucidate additional mutation loci associated with fluoroquinolone (FQ) resistance and evaluate the discriminatory capacity of mutation loci and...
This study aims to elucidate additional mutation loci associated with fluoroquinolone (FQ) resistance and evaluate the discriminatory capacity of mutation loci and allele mutation frequencies in identifying FQ-resistant (MTB) isolates. A random selection of isolates was extracted from an ongoing collection. Drug resistance was determined using the resazurin microtiter assay (REMA) as the gold standard. Mutation loci and the burden of mutations in the quinolone resistance-determining region (QRDR) were elucidated through whole-genome sequencing (WGS). Novel amino acid mutations, namely, G520D and G520T, were identified in the gyrB and associated with FQ resistance. In the context of distinguishing FQ-resistant isolates, the AUC for the QRDR mutation frequency burden (0.969) surpassed that of the mutation locus (0.929), and this difference was statistically significant ( = 0.03). Furthermore, using the resistance mutation locus as a reference, setting the QRDR mutation frequency burden threshold at 1.31% resulted in a 3.60% increase in the accuracy of classifying FQ-resistant isolates (NRI = 3.60%, < 0.001). The QRDR mutation frequency burden appears to offer superior diagnostic efficacy in discriminating FQ-resistant isolates compared to qualitative detection of mutant loci.IMPORTANCEFluoroquinolone (FQ) drugs are recommended as second-line drugs for the treatment of multidrug-resistant tuberculosis. With the massive use of FQ drugs in the clinical treatment of tuberculosis (TB), there is an increasing rate of drug resistance to FQ drugs. In this study, we identified and demonstrated novel amino acid mutations associated with FQ resistance in (MTB), and we quantified the mutation sites and identified the quinolone resistance-determining region (QRDR) mutation frequency burden as a novel diagnostic method for FQ resistance. We hope that the results of this study will provide data support and a theoretical basis for the rapid diagnosis of FQ-resistant MTB.
Topics: Mycobacterium tuberculosis; Fluoroquinolones; Whole Genome Sequencing; Humans; Mutation; Microbial Sensitivity Tests; Antitubercular Agents; Tuberculosis, Multidrug-Resistant; Drug Resistance, Bacterial; Genome, Bacterial; DNA Gyrase
PubMed: 38687077
DOI: 10.1128/spectrum.03930-23 -
Bioinformatics and Biology Insights 2024A variety of active chemicals found in medicinal plants can be used to develop new medications with few adverse effects. In vitro and in silico analyses were used to...
A variety of active chemicals found in medicinal plants can be used to develop new medications with few adverse effects. In vitro and in silico analyses were used to evaluate the anticancer properties of fruit and leaf extracts. Here, we show that the methanolic extract from fruit and leaf extracts inhibits 2 human ovarian cancer cell lines, A2780CP and SKOV-3. The leaf extract demonstrated strong cytotoxicity against A2780CP with an IC50 of 1.2 μg/mL, almost matching the IC50 of the anticancer medication doxorubicin (0.9 μg/mL). Higher antioxidant activity was observed in the fruit than leaf extract. The molecular docking results showed that the active component, podocarpusflavone A, was the best-docked chemical with the human topoisomerase II alpha enzyme. According to our knowledge, this is the first in vitro study to show the cytotoxicity of extracts against the 2 previously described human ovarian cancer cell lines. The fact that the podocarpusflavone A molecule may have an inhibitory effect on the human topoisomerase II alpha enzyme was also revealed by this first in silico analysis. Our findings imply that the fruit and leaf methanolic extract has anticancer characteristics that may guide future in vivo studies.
PubMed: 38681095
DOI: 10.1177/11779322241248904