Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:5.99.1.2 (topoisomerase)
9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Salvicine, a structurally modified diterpenoid quinone derived from Salvia prionitis, is a nonintercalative topoisomerase II (topo II) poison. The compound possesses potent in vitro and in vivo antitumor activity with a broad spectrum of anti-multidrug resistance activity and is currently in phase II clinical trials. To elucidate the distinct antitumor properties of salvicine and obtain valuable structural information of salvicine-topo II interactions, we characterized the effects of salvicine on human topo IIalpha (htopo IIalpha), including possible binding sites and molecular interactions. The enzymatic assays disclosed that salvicine mainly inhibits the catalytic activity with weak DNA cleavage action, in contrast to the classic topo II poison etoposide (VP16). Molecular modeling studies predicted that salvicine binds to the ATP pocket in the ATPase domain and superimposes on the phosphate and ribose groups. In a surface plasmon resonance binding assay, salvicine exhibited higher affinity for the ATPase domain of htopo IIalpha than ATP and ADP. Competitive inhibition tests demonstrated that ATP competitively and dose-dependently blocked the interactions between salvicine and ATPase domain of htopo IIalpha. The data illustrate that salvicine shares a common binding site with ATP and functions as an ATP competitor. To our knowledge, this is the first report to identify an ATP-binding pocket as the structural binding motif for a nonintercalative eukaryotic topo II poison. These findings collectively support the potential value of an ATP competitor of htopo IIalpha in tumor chemotherapy.
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PMID:Salvicine functions as novel topoisomerase II poison by binding to ATP pocket. 1691 42

Overexpression of the HipA protein of the HipBA toxin/antitoxin module leads to multidrug tolerance in Escherichia coli. HipA is a "toxin" that causes reversible dormancy, whereas HipB is an antitoxin that binds HipA and acts as a transcriptional repressor of the hipBA operon. Comparative sequence analysis shows that HipA is a member of the phosphatidylinositol 3/4-kinase superfamily. The kinase activity of HipA was examined. HipA was autophosphorylated in the presence of ATP in vitro, and the purified protein appeared to carry a single phosphate group on serine 150. Thus, HipA is a serine kinase that is at least partially phosphorylated in vivo. Overexpression of HipA caused inhibition of cell growth and increase in persister formation. Replacing conserved aspartate 309 in the conserved kinase active site or aspartate 332 in the Mg2+-binding site with glutamine produced mutant proteins that lost the ability to stop cellular growth upon overexpression. Replacing serine 150 with alanine yielded a similarly inactive protein. The mutant proteins were then examined for their ability to increase antibiotic tolerance. Cells overexpressing wild-type HipA were highly tolerant to cefotaxime, a cell wall synthesis inhibitor, to ofloxacin, a fluoroquinolone inhibitor of DNA gyrase, and to topoisomerase IV and were almost completely resistant to killing by mitomycin C, which forms DNA adducts. The mutant proteins did not protect cells from cefotaxime or ofloxacin and had an impaired ability to protect from mitomycin C. Taken together, these results suggest that the protein kinase activity of HipA is essential for persister formation.
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PMID:Kinase activity of overexpressed HipA is required for growth arrest and multidrug tolerance in Escherichia coli. 1704 Oct 39

Type II DNA topoisomerases are essential and ubiquitous enzymes that perform important functions in chromosome condensation and segregation and in regulating intracellular DNA supercoiling. Topoisomerases carry out these DNA transactions by passing one segment of DNA through the other by using a reversible, enzyme-bridged double strand break. The transient enzyme/DNA adduct is mediated by a phosphodiester bond between the active-site tyrosine and a backbone phosphate of DNA. The opening and closing of the DNA gate, a critical step for strand passage during the catalytic cycle, is coupled to this cleavage/religation. We designed a unique oligonucleotide substrate with a pair of fluorophores straddling the topoisomerase II cleavage site, allowing the use of FRET to monitor the opening of the DNA gate. The DNA substrate undergoes an enzyme-mediated transition between a closed and open state in the presence of ATP, similar to the overall topoisomerase II catalyzed reaction. Single-molecule fluorescence microscopy measurements demonstrate that the transition has comparable rate constants for both the opening and closing reaction during steady-state ATP hydrolysis, with an apparent equilibrium constant near unity. In the presence of AMPPNP, a reduction in FRET occurs, suggesting an opening or partial opening of the DNA gate. However, the single-molecule experiments indicate that the open and closed states do not interconvert at a measurable rate.
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PMID:Single-molecule measurements of the opening and closing of the DNA gate by eukaryotic topoisomerase II. 1736 72

The 2.7 A crystal structure of the 55-kDa N-terminal breakage-reunion domain of topoisomerase (topo) IV subunit A (ParC) from Streptococcus pneumoniae, the first for the quinolone targets from a gram-positive bacterium, has been solved and reveals a 'closed' dimer similar in fold to Escherichia coli DNA gyrase subunit A (GyrA), but distinct from the 'open' gate structure of Escherichia coli ParC. Unlike GyrA whose DNA binding groove is largely positively charged, the DNA binding site of ParC exhibits a distinct pattern of alternating positively and negatively charged regions coincident with the predicted positions of the grooves and phosphate backbone of DNA. Based on the ParC structure, a new induced-fit model for sequence-specific recognition of the gate (G) segment by ParC has been proposed. These features may account for the unique DNA recognition and quinolone targeting properties of pneumococcal type II topoisomerases compared to their gram-negative counterparts.
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PMID:Breakage-reunion domain of Streptococcus pneumoniae topoisomerase IV: crystal structure of a gram-positive quinolone target. 1737 87

The dynamics of the DNA phosphodiester backbone conformations have been studied for a strong topoisomerase II cleavage site (site 22) using molecular dynamics simulations in explicit water and in the presence of sodium ions. We investigated the backbone motions and more particularly the BI/BII transitions involving the epsilon and zeta angles. The consensus cleavage site is adjacent to the phosphate which shows the most important phosphodiester backbone flexibility in the sequence. We infer that these latter properties could be responsible for the preferential cleavage at this site possibly through the perturbation of the cleavage/ligation activities of the topoisomerase II. More generally, the steps pur-pur and pyr-pur are those presenting the highest BII contents. Relations are observed between the backbone phosphodiester BI/BII transitions and the flexibility of the deoxyribose sugar and the helical parameters such as roll. The roll is sequence dependent when the related phosphate is in the BI form, whereas this appears not to be true when it is in the BII form. The BI/BII transitions are associated with water migration, and new relations are observed with counterions. Indeed, it is observed that a strong coupling exists between the BII form and the presence of sodium ions near the adjacent sugar deoxyribose. The presence of sodium ions in the O4' surroundings or their binding could assist the BI to BII transition by furnishing energy. The implications of these new findings and, namely, their importance in the context of the sequence-dependent behavior of BI/BII transitions will be investigated in future studies.
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PMID:Conformations and dynamics of the phosphodiester backbone of a DNA fragment that bears a strong topoisomerase II cleavage site. 1739 Oct 20

A series of nitrofuran-based compounds were identified as inhibitors of estrogen signaling in a cell-based, high-throughput screen of a diverse library of small molecules. These highly related compounds were subsequently found to inhibit topoisomerase II in vitro at concentrations similar to that required for the inhibition of estrogen signaling in cells. The most potent nitrofuran discovered is approximately 10-fold more active than etoposide phosphate, a topoisomerase II inhibitor in clinical use. The nitrofurans also inhibit topoisomerase I activity, with approximately 20-fold less activity. Moreover, the nitrofurans, in contrast to etoposide, induce a profound cell cycle arrest in the G(0)-G(1) phase of the cell cycle, do not induce double-stranded DNA breaks, are not substrates for multidrug resistance protein-1 export from the cell, and are amenable to synthetic development. In addition, the nitrofurans synergize with etoposide phosphate in cell killing. Clonogenic assays done on a panel of human tumors maintained ex vivo in nude mice show that the most active compound identified in the screen is selective against tumors compared with normal hematopoietic stem cells. However, this compound had only moderate activity in a mouse xenograft model. This novel class of topoisomerase II inhibitor may provide additional chemotherapeutic strategies for the development of cytotoxic agents with proven clinical utility.
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PMID:Thanatop: a novel 5-nitrofuran that is a highly active, cell-permeable inhibitor of topoisomerase II. 1748 60

Human polynucleotide kinase (hPNK) is a bifunctional enzyme possessing a 5'-DNA kinase activity and a 3'-phosphatase activity. Studies based on cell extracts and purified proteins have indicated that hPNK can act on single-strand breaks and double-strand breaks (DSB) to restore the termini to the chemical form required for further action by DNA repair polymerases and ligases (i.e., 5'-phosphate and 3'-hydroxyl termini). These studies have revealed that hPNK can bind to XRCC4, and as a result, hPNK has been implicated as a participant in the nonhomologous end joining (NHEJ) pathway for DSB repair. We sought to confirm the role of hPNK in NHEJ in the cellular setting using a genetic approach. hPNK was stably down-regulated by RNA interference expression in M059K glioblastoma cells, which are NHEJ positive, and M059J cells, which are NHEJ deficient due to a lack of DNA-PK catalytic subunit (DNA-PKcs). Whereas depletion of hPNK significantly sensitized M059K cells to ionizing radiation, no additional sensitization was conferred to M059J cells, clearly implying that hPNK operates in the same DNA repair pathway as DNA-PKcs. On the other hand, depletion of hPNK did not increase the level of sister chromatid exchanges, indicating that hPNK is not involved in the homologous recombination DSB repair pathway. We also provide evidence that the action of hPNK in the repair of camptothecin-induced topoisomerase 1 "dead-end" complexes is independent of DNA-PKcs and that hPNK is not involved in the nucleotide excision repair pathway.
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PMID:Human polynucleotide kinase participates in repair of DNA double-strand breaks by nonhomologous end joining but not homologous recombination. 1763 72

9-nitro-20(S)-camptothecin (9-NC) is a potent topoisomerase-I inhibitor, and it was applied for clinical trials in cancer treatment. However, the applications of 9-NC were limited by its poor solubility and instability. In order to overcome these disadvantages, 9-NC was encapsulated in amphiphilic copolymer micelles composed of methoxy poly(ethylene glycol)-b-poly(D,L-lactide) (mPEG-PDLLA, PELA). Three diblock copolymers with different PDLLA chain lengths were synthesized. The critical micelle concentration was varied from 10(-4) g L(-1) to 10(-2) g L(-1). The 9-NC loaded micelles were nanospheres with diameters ranging from 30 nm to 60 nm. The relationship between the composition of copolymers and the drug loading content was discussed. The encapsulation of micelles improved the solubility of 9-NC greatly. The solubility of 9-NC in micelle M1 was about 250 times higher than that of 9-NC in a phosphate buffer solution (PBS). The stability of 9-NC in micelles was also promoted. After being incubated in PBS for 160 min, 80% of 9-NC in micelles existed as an active lactone form, while 85% of 9-NC in PBS were transferred to an inactive carboxylate salt form. The release experiments were carried out in PBS and the results showed that the release processes were controllable.
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PMID:Controlled release of 9-nitro-20(S)-camptothecin from methoxy poly(ethylene glycol)-poly(D,L-lactide) micelles. 1845

Human topoisomerase IB (hTopo) forms a covalent phosphotyrosyl linkage with the DNA backbone, and controls genomic DNA topology by relaxing DNA supercoils during the processes of DNA replication, transcription, chromosome condensation and decondensation. The essential role of hTopo in these processes has made it a preeminent anticancer drug target. We have screened a small library of arylstibonic acids for their effects on plasmid supercoil relaxation catalyzed by hTopo. Despite the similar structures of the library compounds, some compounds were found to be effective competitive inhibitors, and others, nonessential activators. Some arylstibonic acids show selectivity in their action against hTopo and the related enzyme from poxvirus (vTopo). Structure-activity relationships and structural modeling suggest that competitive inhibition may result from positioning of the negatively charged stibonic acid and carboxylate groups of the inhibitors into DNA phosphate binding pockets on hTopo. The hTopo activators act by a surprising allosteric mechanism without interfering with DNA binding or binding of the widely used hTopo poison camptothecin. Arylstibonic acid competitive inhibitors may become useful small molecules for elucidating the cellular functions of hTopo.
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PMID:Arylstibonic acids: novel inhibitors and activators of human topoisomerase IB. 1850 7

The DNA cleavage reaction of human topoisomerase IIalpha is critical to all of the physiological and pharmacological functions of the protein. While it has long been known that the type II enzyme requires a divalent metal ion in order to cleave DNA, the role of the cation in this process is not known. To resolve this fundamental issue, the present study utilized a series of divalent metal ions with varying thiophilicities in conjunction with DNA cleavage substrates that replaced the 3'-bridging oxygen of the scissile bond with a sulfur atom (i.e. 3'-bridging phosphorothiolates). Rates and levels of DNA scission were greatly enhanced when thiophilic metal ions were included in reactions that utilized sulfur-containing substrates. Based on these results and those of reactions that employed divalent cation mixtures, we propose that topoisomerase IIalpha mediates DNA cleavage via a two-metal-ion mechanism. In this model, one of the metal ions makes a critical interaction with the 3'-bridging atom of the scissile phosphate. This interaction greatly accelerates rates of enzyme-mediated DNA cleavage, and most likely is needed to stabilize the leaving 3'-oxygen.
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PMID:Human topoisomerase IIalpha uses a two-metal-ion mechanism for DNA cleavage. 1865 31


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