Increased sensitivity to quinolone antibacterials can be engineered in human topoisomerase IIalpha by selective mutagenesis.

A potential region of drug-DNA interaction in the A subunit of DNA gyrase has previously been identified from crystallographic studies. The local amino acid sequence has been compared with similar regions in yeast topoisomerase II and human topoisomerase IIalpha. Three non- conserved, potentially solvent-accessible residues at positions 762, 763 and 766 in human topoisomerase IIalpha lie between well-conserved regions. The corresponding residues in GyrA (83, 84 and 87) have a high frequency of mutation in quinolone-resistant bacteria. Mutations in human topoisomerase IIalpha have been generated in an attempt to engineer ciprofloxacin sensitivity into this enzyme: M762S, S763A and M766D (each mutated to the identical amino acid present in gyrase), along with an M762S/S763A double mutant and a triple mutant. These enzymes were introduced into a temperature-sensitive yeast strain, deficient in topoisomerase II, for in vivo studies, and were overproduced for in vitro studies. The M766D mutation renders the enzyme incapable of supporting the temperature-sensitive strain at a non-permissive temperature. However, both M766D and the triple mutant enzymes can be overproduced and are fully active in vitro. The double mutant was impaired in its ability to cleave DNA and had reduced catalytic activity. The triple mutation confers a three-fold increase in sensitivity to ciprofloxacin in vitro and similar sensitivities to a range of other quinolones. The activity of the quinolone CP-115,953, a bacterial and eukaryotic topoisomerase II poison, was unaffected by any of these mutations. Mutations in this region were found to increase the sensitivity of the enzyme to the DNA intercalating anti-tumour agents m-AMSA and ellipticine, but confer resistance to the non-intercalating agents etoposide, teniposide and merbarone, an effect that was maximal in the triple mutant. We have therefore shown the importance of this region in determining the sensitivity of topoisomerase II to drugs and have engineered increased sensitivity to quinolones.

Antitumor polycyclic acridines. 7. Synthesis and biological properties of DNA affinic tetra- and pentacyclic acridines.

New synthetic routes to a series of tetra- and pentacyclic acridines related in structure to marine natural products are reported. The novel water-soluble agent dihydroindolizino[7,6,5-kl]acridinium chloride 14 has inhibitory activity in a panel of non-small-cell lung and breast tumor cell lines exceeding that of m-AMSA. The salt inhibited the release of minicircle products of kDNA confirming that disorganization of topoisomerase II partly underlies the activity of the compound. COMPARE analysis of the NCI mean graph profile of compound 14 at the GI(50) level corroborates this conclusion with Pearson correlation coefficients (>0.6) to clinical agents of the topoisomerase II class: however, this correlation was not seen at the LC(50) level. The inhibitory action of 14 on Saccharomyces cerevisiae transfected with human topoisomerase II isoforms showed a 3-fold selectivity against the IIalpha isoform over the IIbeta isoform. Unlike m-AMSA, 14 is not susceptible to P-glycoprotein-mediated drug efflux and retains activity in lung cells with derived resistance to the topoisomerase II inhibitor etoposide.

The N-terminal domain of human topoisomerase IIalpha is a DNA-dependent ATPase.

We have constructed clones encoding N-terminal fragments of human DNA topoisomerase IIalpha. We show that the N-terminal domain (approximately 50 kDa) has an intrinsic ATPase activity that can be stimulated by DNA. The enzyme obeys Michaelis-Menten kinetics showing a approximately 6-fold increase in kcat in the presence of DNA. Cross-linking studies indicate that the N-terminal domain is a dimer in the absence and presence of nucleotides. Using site-directed mutagenesis, we have identified the catalytic residue for ATP hydrolysis as Glu86. Phosphorylation of the N-terminal domain with protein kinase C does not affect the ATPase activity. The ATPase domain of human topoisomerase IIalpha shows significant differences from its counterpart in DNA gyrase and we discuss the mechanistic implications of these data.

Use of a rapid throughput in vivo screen to investigate inhibitors of eukaryotic topoisomerase II enzymes.

Topoisomerase II catalyzes the passage of one DNA helix through another via a transient double-stranded break. The essential nature of this enzyme in cell proliferation and its mechanism of action make it an ideal target for cytotoxic agents. Saccharomyces cerevisiae topoisomerase II has been frequently used as a model for testing potential inhibitors of eukaryotic topoisomerase II as antitumor agents. The standard in vivo method of estimating the sensitivity of S. cerevisiae to the antitopoisomerase drugs is via inhibition or kill curves which rely on viable-cell counts and is labor intensive. We present an alternative to this, a high-throughput in vivo screen. This method makes use of a drug-permeable S. cerevisiae strain lacking endogenous topoisomerase II, which is modified to express either human topoisomerase IIalpha or IIbeta or S. cerevisiae topoisomerase II carried on plasmids. Each modified strain expresses a full-length topoisomerase II enzyme, as opposed to the more commonly used temperature-sensitive S. cerevisiae mutant expressing yeast or yeast/human hybrid enzymes. A comparison of this new method with a plating-and-counting method gave similar drug sensitivity results, with increased accuracy and reduced manual input for the new method. The information generated has highlighted the sensitivities of different topoisomerase II enzymes and isoenzymes to several different classes of topoisomerase II inhibitor.

The DNA dependence of the ATPase activity of human DNA topoisomerase IIalpha.

We have purified human topoisomerase IIalpha from HeLa cells and studied its ATPase reaction. The ATPase activity is stimulated by DNA and shows apparent Michaelis-Menten kinetics. Although the ATPase activity of human topoisomerase IIalpha is lower than that of Saccharomyces cerevisiae, it is more active in decatenation, implying more efficient coupling of the ATPase to DNA strand passage under these conditions. Using plasmid pBR322 as the DNA cofactor, the reaction shows hyperstimulation by DNA at a base pair to enzyme dimer ratio of 100-200:1. When DNA fragments are used as the cofactor, the reaction requires > approximately 100 base pairs to stimulate the activity and fragments of approximately 300 base pairs show hyperstimulation. This behavior can be rationalized in terms of the enzyme requiring fragments that can bind to both the DNA gate and the ATP-operated clamp in order for the ATPase reaction to be stimulated. Hyperstimulation is a consequence of the saturation of DNA with enzyme. The mechanistic implications of these results are discussed.

Studies to show that with podophyllotoxin the early replicative stages of herpes simplex virus type 1 depend upon functional cytoplasmic microtubules.

The antiviral activity of podophyllotoxin against herpes simplex type 1 virus (HSV-1) grown in Vero cells was studied by a simple microtitration assay. Antiviral effects were induced at similar concentrations as direct cellular toxicity, as characterised by a time-dependent loss of cell monolayer. Podophyllotoxin-mediated toxicity arises from cytoplasmic microtubular, and hence cytoskeletal, decay. Some degree of selectivity was seen for inhibition of virus replication over direct cellular toxicity. Podophyllotoxin acted against an early viral process, as an antiviral effect was still seen if drug was removed 2 h after infection. Similar effects were seen with colchicine, a classical tubulin-binding compound, but not with bromovinyldeoxyuridine. Podophyllotoxin was capable of inducing a cytoprotective effect in Vero cells, as pre-treatment of cells abrogated virus growth for up to 90 min after removal of drug. This is coincident with the repolymerisation of cellular microtubules and re-formation of the cytoskeleton. We conclude that HSV-1 relies upon a functional cellular cytoskeleton for efficient completion of an early replicative event. Such a process may be the transport of viral material to the nucleus or inhibition of the formation of intranuclear viral 'replication factories', bodies containing cytoskeletal fragments constructed after viral infection.