An in vitro DNA relaxation assay is often used to measure TopI activity. TopI is known to relax supercoiled plasmid DNA to an open circular form in vitro and in vivo. Here CPT inhibition of supercoiled DNA relaxation in vitro was evaluated. Recombinant hTopIs induction of supercoiled pUC19 plasmid novel relax ation was used as the assay system, and the results are shown in Figure 2B. Because of their different densities, supercoiled DNA migrated faster on the agarose gel than did relaxed circular DNA shown in the control. CPT treatment inhibited TopI relaxation activity, and a greater amount of uncatalytic supercoiled DNA was retained in a concentration dependent manner. The results ensure the avail ability of all materials, including purified recombinant hTopI, the pUC19 plasmid, and CPT, for subsequent assays of TopI catalysis.

SPR assay of covalent complex formation The SPR assay was used to measure the formation of the DNA TopI cleavage complex. This assay differs from the gel assay by its high throughput, being in real time and label free, and directly determining Inhibitors,Modulators,Libraries the binding between the analyte and ligand. Recombinant hTopI was cova lently coupled to Inhibitors,Modulators,Libraries the carboxylmethylated dextran surface of the chip using standard amine coupling chemistry. The immobilization curves are shown in Figure 3A. The highest level of immobilization was achieved at 4000 RU. The binding of anti hTopI antibodies to immobilized hTopI was observed in real time after reference subtrac tion of the response of the hTopI free control. The response was proportional to the antibody concentration while the signals were fairly weak in the hTopI free channel.

The pUC19 plas mid was loaded onto the hTopI immobilized sensor chip, and binding affinities were analyzed. The Inhibitors,Modulators,Libraries binding of the pUC19 plasmid to immobilized hTopI was detected by the concentration dependent increase in RU, which suggests Inhibitors,Modulators,Libraries that the sensor chip immobilized hTopI retained its DNA binding activity. RU values of CPT alone in the analyte flowing through the sen sor chip remained fairly constant, which indicates that CPT did not bind to hTopI without DNA. This suggests that the binding of CPT to TopI on the sensor chip was dependent on the DNA content because CPT bound to hTopI at the stage of forming intermediates of the TopI DNA cleavage complex. To characterize the drug binding kinetics using the SPR sen sor chip, plasmid DNA was included in the analyte.

The combination of pUC19 plasmid DNA and CPT as the analyte was measured flowing through the sensor chip, and the RU increased in a con centration dependent Inhibitors,Modulators,Libraries manner with a KD value of 4. 1 10 29 compared to DNA only, according to the ProteOn Man ager 2. 0 calculation. Gemcitabine supplier In the presence of the TopI inhibitor, CPT, re ligation was impeded. and DNA and TopI were trapped in a covalent cleavage complex. Similar results were obtained with a different TopI inhibitor, EVO, with a KD value of 5.