Inhibition of archaeal growth and DNA topoisomerase VI activities by the Hsp90 inhibitor radicicol

Type II DNA topoisomerases have been classified into two families, Topo IIA and Topo IIB, based on structural and mechanistic dissimilarities. Topo IIA is the target of many important antibiotics and antitumoural drugs, most of them being inactive on Topo IIB. The effects and mode of action of Topo IIA inhibitors in vitro and in vivo have been extensively studied for the last twenty-five years. In contrast, studies of Topo IIB inhibitors were lacking. To document this field, we have studied two Hsp90 inhibitors (radicicol and geldanamycin), known to interact with the ATP-binding site of Hsp90 (the Bergerat fold), which is also present in Topo IIB. Here, we report that radicicol inhibits the decatenation and relaxation activities of Sulfolobus shibatae DNA topoisomerase VI (a Topo IIB) while geldanamycin does not. In addition, radicicol has no effect on the Topo IIA Escherichia coli DNA gyrase. In agreement with their different effects on DNA topoisomerase VI, we found that radicicol can theoretically fit in the ATP-binding pocket of the DNA topoisomerase VI ‘Bergerat fold’, whereas geldanamycin cannot. Radicicol inhibited growths of Sulfolobus acidocaldarius (a crenarchaeon) and of Haloferax volcanii (a euryarchaeon) at the same doses that inhibited DNA topoisomerase VI in vitro. In contrast, the bacteria E.coli was resistant to this drug. Radicicol thus appears to be a very promising compound to study the mechanism of Topo IIB in vitro, as well as the biological roles of these enzymes in vivo

topIb, a phylogenetic hallmark gene of Thaumarchaeota encodes a functional eukaryote-like topoisomerase IB

International audienceType IB DNA topoisomerases can eliminate torsional stresses produced during replication and transcription. These enzymes are found in all eukaryotes and a short version is present in some bacteria and viruses. Among prokaryotes, the long eukaryotic version is only observed in archaea of the phylum Thau-marchaeota. However, the activities and the roles of these topoisomerases have remained an open question. Here, we demonstrate that all available thaumar-chaeal genomes contain a topoisomerase IB gene that defines a monophyletic group closely related to the eukaryotic enzymes. We show that the topIB gene is expressed in the model thaumarchaeon Ni-trososphaera viennensis and we purified the recom-binant enzyme from the uncultivated thaumarchaeon Candidatus Caldiarchaeum subterraneum. This enzyme is active in vitro at high temperature, making it the first thermophilic topoisomerase IB characterized so far. We have compared this archaeal type IB enzyme to its human mitochondrial and nuclear counterparts. The archaeal enzyme relaxes both negatively and positively supercoiled DNA like the eukaryotic enzymes. However, its pattern of DNA cleavage specificity is different and it is resistant to camptothecins (CPTs) and non-CPT Top1 inhibitors, LMP744 and lamellarin D. This newly described ther-mostable topoisomerases IB should be a promising new model for evolutionary, mechanistic and structural studies

Structural basis for topoisomerase VI inhibition by the anti-Hsp90 drug radicicol

Members of the GHL ATPase superfamily, including type II topoisomerases, Hsp90-class chaperones, and MutL, all share a common GHKL-type ATP-binding fold and act as nucleotide-controlled ‘molecular clamps’. These enzymes' ATP-binding sites have proven to be rich drug targets, and certain inhibitors of type II topoisomerases and Hsp90 bind to this region and competitively inhibit these enzymes. Recently, it was found that radicicol, a drug known to block Hsp90 function, also inhibits the archaeal type IIB topoisomerase topo VI. Here, we use X-ray crystallography to show that despite low sequence identity (∼10–12%) between topo VI and Hsp90, radicicol binds to the ATPase sites of these two enzymes in an equivalent manner. We further demonstrate that radicicol inhibits both the dimerization of the topo VI ATPase domains and ATP hydrolysis, two critical steps in the enzyme's strand passage reaction. This work contributes to a growing set of structures detailing the interactions between GHL-family proteins and various drugs, and reveals radicicol as a versatile scaffold for targeting distantly related GHL enzymes

Amphiphilic block copolymers from a renewable Ɛ-decalactone monomer: prediction and characterization of micellar core effects on drug encapsulation and release

Here we describe a methoxy poly(ethyleneglycol)-b-poly(ε-decalactone) (mPEG-b-PεDL) copolymer and investigate the potential of the copolymer as a vehicle for solubilisation and sustained release of indomethacin (IND). The indomethacin loading and release from mPEG-b-PεDL micelles (amorphous cores) was compared against methoxy poly(ethyleneglycol)-b-poly(ε-caprolactone)(mPEG-b-PCL) micelles (semicrystalline cores). The drug–polymer compatibility was determined through a theoretical approach to predict drug incorporation into hydrated micelles. Polymer micelles were prepared by solvent evaporation and characterised for size, morphology, indomethacin loading and release. All the formulations generated spherical micelles but significantly larger mPEG-b-PεDL micelles were observed compared to mPEG-b-PCL micelles. A higher compatibility of the drug was predicted for PCL cores based on Flory–Huggins interaction parameters (χsp) using the Hansen solubility parameter (HSP) approach, but higher measured drug loadings were found in micelles with PεDL cores compared to PCL cores. This we attribute to the higher amorphous content in the PεDL-rich regions which generated higher micellar core volumes. Drug release studies showed that the semicrystalline PCL core was able to release IND over a longer period (80% drug release in 110 h) compared to PεDL core micelles (80% drug release in 72 h)

The key DNA-binding residues in the C-terminal domain of Mycobacterium tuberculosis DNA gyrase A subunit (GyrA)

As only the type II topoisomerase is capable of introducing negative supercoiling, DNA gyrase is involved in crucial cellular processes. Although the other domains of DNA gyrase are better understood, the mechanism of DNA binding by the C-terminal domain of the DNA gyrase A subunit (GyrA-CTD) is less clear. Here, we investigated the DNA-binding sites in the GyrA-CTD of Mycobacterium tuberculosis gyrase through site-directed mutagenesis. The results show that Y577, R691 and R745 are among the key DNA-binding residues in M.tuberculosis GyrA-CTD, and that the third blade of the GyrA-CTD is the main DNA-binding region in M.tuberculosis DNA gyrase. The substitutions of Y577A, D669A, R691A, R745A and G729W led to the loss of supercoiling and relaxation activities, although they had a little effect on the drug-dependent DNA cleavage and decatenation activities, and had no effect on the ATPase activity. Taken together, these results showed that the GyrA-CTD is essential to DNA gyrase of M.tuberculosis, and promote the idea that the M.tuberculosis GyrA-CTD is a new potential target for drug design. It is the first time that the DNA-binding sites in GyrA-CTD have been identified

Detection of changes in gene regulatory patterns, elicited by perturbations of the Hsp90 molecular chaperone complex, by visualizing multiple experiments with an animation

<p>Abstract</p> <p>Background</p> <p>To make sense out of gene expression profiles, such analyses must be pushed beyond the mere listing of affected genes. For example, if a group of genes persistently display similar changes in expression levels under particular experimental conditions, and the proteins encoded by these genes interact and function in the same cellular compartments, this could be taken as very strong indicators for co-regulated protein complexes. One of the key requirements is having appropriate tools to detect such regulatory patterns.</p> <p>Results</p> <p>We have analyzed the global adaptations in gene expression patterns in the budding yeast when the Hsp90 molecular chaperone complex is perturbed either pharmacologically or genetically. We integrated these results with publicly accessible expression, protein-protein interaction and intracellular localization data. But most importantly, all experimental conditions were simultaneously and dynamically visualized with an animation. This critically facilitated the detection of patterns of gene expression changes that suggested underlying regulatory networks that a standard analysis by pairwise comparison and clustering could not have revealed.</p> <p>Conclusions</p> <p>The results of the animation-assisted detection of changes in gene regulatory patterns make predictions about the potential roles of Hsp90 and its co-chaperone p23 in regulating whole sets of genes. The simultaneous dynamic visualization of microarray experiments, represented in networks built by integrating one's own experimental with publicly accessible data, represents a powerful discovery tool that allows the generation of new interpretations and hypotheses.</p

Structural Insights into the Quinolone Resistance Mechanism of Mycobacterium tuberculosis DNA Gyrase

Mycobacterium tuberculosis DNA gyrase, an indispensable nanomachine involved in the regulation of DNA topology, is the only type II topoisomerase present in this organism and is hence the sole target for quinolone action, a crucial drug active against multidrug-resistant tuberculosis. To understand at an atomic level the quinolone resistance mechanism, which emerges in extensively drug resistant tuberculosis, we performed combined functional, biophysical and structural studies of the two individual domains constituting the catalytic DNA gyrase reaction core, namely the Toprim and the breakage-reunion domains. This allowed us to produce a model of the catalytic reaction core in complex with DNA and a quinolone molecule, identifying original mechanistic properties of quinolone binding and clarifying the relationships between amino acid mutations and resistance phenotype of M. tuberculosis DNA gyrase. These results are compatible with our previous studies on quinolone resistance. Interestingly, the structure of the entire breakage-reunion domain revealed a new interaction, in which the Quinolone-Binding Pocket (QBP) is blocked by the N-terminal helix of a symmetry-related molecule. This interaction provides useful starting points for designing peptide based inhibitors that target DNA gyrase to prevent its binding to DNA

The ancestral role of ATP hydrolysis in type II topoisomerases: prevention of DNA double-strand breaks

Type II DNA topoisomerases (topos) catalyse changes in DNA topology by passing one double-stranded DNA segment through another. This reaction is essential to processes such as replication and transcription, but carries with it the inherent danger of permanent double-strand break (DSB) formation. All type II topos hydrolyse ATP during their reactions; however, only DNA gyrase is able to harness the free energy of hydrolysis to drive DNA supercoiling, an energetically unfavourable process. A long-standing puzzle has been to understand why the majority of type II enzymes consume ATP to support reactions that do not require a net energy input. While certain type II topos are known to ‘simplify’ distributions of DNA topoisomers below thermodynamic equilibrium levels, the energy required for this process is very low, suggesting that this behaviour is not the principal reason for ATP hydrolysis. Instead, we propose that the energy of ATP hydrolysis is needed to control the separation of protein–protein interfaces and prevent the accidental formation of potentially mutagenic or cytotoxic DSBs. This interpretation has parallels with the actions of a variety of molecular machines that catalyse the conformational rearrangement of biological macromolecules