WO2009037510A1 - Dna decatenation assay 997 - Google Patents

Abstract

The present invention provides methods for assaying the activity of DNA decatenation enzymes and methods for screening for compounds that modulate the activity of DNA decatenation enzymes.

Description

102997

DNA DECATENATION ASSAY 997

FIELD OF THE INVENTION The present invention relates to methods for assaying DNA Decatenation and methods for identifying compounds that modulate the DNA decatenation function of topoisomerase.

BACKGROUND

DNA topoisomerases all share the property of catalyzing interconversion between different topological forms of DNA. DNA topoisomerases have been isolated from viral, prokaryotic, and eukaryotic sources. There are two classes of topoisomerase enzymes (termed type I and type II) that are distinguished by an operational difference; the type I enzymes catalyze DNA interconversion during which the linking number changes in steps of one, while the type II enzymes perform reactions during which the linking number changes in steps of two. Negatively supercoiled DNA is more easily unwound, allowing RNA polymerase to bind more readily to the DNA, hence promoting the transcription of certain genes (Reece & Maxwell, 1991, Crit. Rev. Biochem. MoI. Biol, 26:335-375).

Assays of topoisomerase activity are important for the identification of compounds that modulate topoisomerase activity. Topoisomerases in bacteria are targets for antibacterial agents while topoisomerases in eukaryotes are targets for anti-cancer drugs. In bacteria, type II topoisomerases include topoisomerase II and topoisomerase IV. The major function of topoisomerase II is DNA supercoiling. The major function of Topoisomerase IV is DNA decatenation. In addition, Topoisomerase II also has the DNA decatenation activity. Topoisomerase IV has DNA relaxation activity. In human, topoisomerase II, like bacterial topoisomerase IV, has DNA decatenation and DNA relaxation activities. DNA decatenation proteins are known infection and cancer drug targets [K.D. Corbett et al. Annu. Rev. Biophys. Biomol. Struct, 33, 95-118 (2004)]

Typically assays for identifying compounds that modulate the DNA decatenation activity are gel-based. These types of assays have many drawbacks including being labour intensive because multiple low throughput steps are involved throughout the process including setting the assay up, acquiring the data and processing the data. Gel-based assays are also error prone in 102997

quantitation due to the fact that the band intensity of DNA in the gel depends on a number of factors. Thus, there exists a need for a DNA decatenation assay, which is quick and accurate.

SUMMARY

The present invention provides a method for identifying compounds that inhibit the DNA decatenation function of a topoisomerase. The method includes determining the effect of a test compound on a decantenation enzyme by contacting a concatenated DNA, a decantenation enzyme and a test compound; separating the concatenated DNA from the decatenated

DNA using a filter; and detecting the presence of filtered decatenated DNA wherein the reduced amount or absence of decatenated DNA is indicative that the test compound inhibits the activity of the decantenation enzyme compared to a control (for example, no test compound). In one embodiment, the decantenation enzyme is a topoisomerase. The topoisomerase can be a prokaryotic topoisomerase IV or a eukaryotic topoisomerase II. The presence of decatenated DNA can be detected by any method including using an agent that binds to the decatenated DNA. In one embodiment, the detecting agent is a fluorescent dye of SYBR Green II, RiboGreen, and YO-PRO-I. The filter that is used to separate the decatenated DNA from the concatenated DNA can be a microfϊlter.

DETAILED DESCRIPTION

The present invention provides a method of screening for compounds that modulate the activity of a topoisomerase. Specifically, compounds can be identified by the methods of the invention that inhibit the DNA decatenation function of a topoisomerase. The assay of the present invention is a solution based method and provides an efficient means to perform a throughput assay for identifying compounds that inhibit the DNA decatenation activity of topoisomerases. Specifically the invention includes contacting a concatenated DNA, a decatenation enzyme and a test compound; separating the concatenated DNA from the decatenated DNA using a filter; and detecting the presence of the filtered decatenated DNA wherein the reduced amount or absence of decatenated DNA is indicative that the test compound inhibits the activity of the decantenation enzyme. By utilizing a filter that can readily separate concatenated DNA from decatenated DNA, the method of the invention provides a quick and easy means of identifying a compound that modulates the DNA decatenation 102997

function of a topoisomerase. The use of a filter has the further advantage of eliminating the need to use any type of solid phase to facilitate detection of the decantenated product. Since the assays of the present invention do not depend on complicated inter-phase interactions or coupled functions, they provide highly specific measurement of DNA decatenation activity. Assays of the present invention are amenable to medium and high-throughput screening

(HTS) formats.

The Decatenation Enzyme

Any agent, such as any topoisomerase that has decatenation activity, can be used in the assays of the present invention. Topoisomerases have been identified in prokaryotes and eukaryotes. Examples of suitable topoisomerases include topoisomerase VI from bacteria and topoisomerase II from eukaryotes. The topoisomerase VI from bacteria can be from a gram positive or gram negative bacterium. Examples of bacteria include Haemophilus influenzae, Moraxella catarrhalis, Pseudomonas aeruginosa, Escherichia coli, Chlamydia spp, Legionella spp, Staphylococcus aureus, Staphylococcus saprophyticus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus mutans, Enterococcus faecalis, Enterococcus faecium, Mycoplasma spp, Bacteroides spp and Clostridium spp. Both bacterial and human topoisomerases with DNA decatenation activity are useful for drug discovery in the infection and cancer areas. The bacterial enzymes are the drug targets for infection. The human enzyme is the drug target for cancer.

A DNA decatenation enzyme can be obtained for use in the present invention according to procedures well known to those in the art. A DNA decatenation enzyme can be obtained by isolation or purification from natural sources or can be expressed using recombinant technology. The enzyme can be expressed as a single target protein or co-expressed with other proteins. The enzyme can be expressed with or without peptide tags or fusion proteins. The enzyme can be isolated as a cell extract, prepared in substantially pure form as a single protein, or prepared as a protein complex. Numerous techniques for obtaining the DNA decatenation enzyme proteins, including bacterial DNA decatenation enzyme proteins, have been described in the literature. See for example, H. Peng et al. J. Biol. Chem. 268, 24481-24490 (1993); M.H. Barnes et al, Protein Expression and Purification, 29, 259-264 (2003); X. Pan et al. Antimicrobial Agents and Chemotherapy, 1129-1136 (1999). Catalytically active portions or fragments of the DNA 102997

decatenation enzyme can also be used in the assays of the present invention. See for example, S. Bellon et al. Antimicrobial Agents and Chemotherapy, 1856-1864 (2004)

A Concatenated DNA The method includes providing a concatenated form of a DNA. The concatenated DNA will typically be double stranded such as a double stranded plasmid or a condensed genomic DNA. Typically concatenated DNA will be larger than 25,000 bp but any appropriate sized DNA can be used as long as separation of concatenated/decatenated DNA can be achieved using a filter of choice. Concatenated DNA for use in the present invention can be bought commercially, for example from Topogen, Inc. (Florida) or can be made by recombinant means. See for example, S. Dasgupta et al. Analytical Biochemistry, 158, 189-194 (1986) and T. A. Shapiro et al, DNA Topoisomerase Protocols Vol. I, Human Press Inc., N. Jersey, pp61-68 (1999).

Detecting Decatenated DNA

To determine if a test compound inhibits the activity of the decatenation enzyme, the reaction mix of the decatenation enzyme, the concatenated DNA and the test compound are passed through a filter that can separate the concatenated DNA from the decatenated DNA. Any filter can be chosen as long as it allows for the separation of concatenated/decatenated DNA. Typically a filter can be chosen based on pore size (physical diameter) or molecular weight. In one example, the filter can have a pore size of less than lμM, e.g., 0.8, 0.6, 0.4, 0.2 μM. In another example, the filter can be a filter that separates molecules of more than 100 KD. An example of a filter that can be used in the method of the invention is a microplate filter.

The filtered decatenated DNA can be detected by any means known to someone skilled in the art. For example, the decatenated DNA can be contacted with a DNA binding dye, such as an intercalating dye or a fluorescent dye including acridine orange and its dimer, 7-AAD, actinomycin D, ACMA, DAPI, dihydroethidium, ethidium bromide and its dimer, hexidium iodide, Hoechst series, hydroxystilbamidine, LDS 751, nuclear yellow, SYBR Green series, PicoGreen, OliGreen, YO-PRO and YOYO series, LO-PRO and LOLO series, BO-PRO and BOBO series, TO-Pro and TOTO series and propidium iodide. Commercially available fluorescent dyes can be found in a Molecular Probes catalog. See specifically Handbook of 102997

Fluorescent Probes and Research Products, by R. P. Haugland, Molecular Probes, Ninth Edition (2002). In one example, the dyes can be selected from the group of RiboGreen, SYBR Green II, and YO-PRO-I. Fluorescent signals and intensity can be detected with standard fluorescence readers or scanners known to the art. The fluorescent dye can be included during the enzymatic reaction or it can be added after.

Screening for Compounds that inhibit the Decatenated Enzyme

The modulator screening assays comprise combining, in the presence and in the absence of a test compound, a concatenated DNA, a DNA decatenation enzyme, and a nucleoside triphosphate under conditions in which the DNA decatenation enzyme is catalytically active and decatenates the concatenated DNA. The product of the reaction is then filtered and the presence of decatenated DNA in the filtered product is determined for example by adding a fluorescent dye that binds the DNA. The fluorescent signal detected in the presence of the test compound is compared to the fluorescent signal detected in the absence of the test compound. A reduction or absence of a fluorescent signal detected in the presence of the compound in the filtered product compared to the fluorescent signal detected in the absence of the compound is indicative of a compound that inhibits DNA decatenation enzyme activity.

Assays of the present invention are conducted under conditions such that the DNA decatenation enzyme is catalytically active. These are conditions under which the DNA decatenation enzyme is capable of decatenation of the concatenated DNA in the presence of a nucleoside triphosphates such as ATP. Such conditions are known to those of skill in the art. A wide variety of incubation conditions can be used, depending on the enzyme used. Reaction conditions in which DNA decatenation enzyme is active in vitro are known in the art. Assays of the present invention can be performed in different formats. In some embodiments, a high-throughput screening (HTS) format is used. HTS can be run at different temperatures, but it is important to keep a constant temperature for the reaction and controls. In some embodiments, room temperature is used.

Nucleoside triphosphates used in the assays of the present invention can be in modified or unmodified forms, including, but not limited to, radioactively labeled or fluorescence-labeled, or modified for activity or detection purposes. 102997

The invention is further illustrated by way of the following examples, which are intended to elaborate several embodiments of the invention. These examples are not intended to, nor are they to be construed to, limit the scope of the invention. It will be clear that the invention may be practiced otherwise than as particularly described herein. Numerous modifications and variations of the present invention are possible in view of the teachings herein and, therefore, are within the scope of the invention.

EXAMPLES Example 1 A DNA decatenation reaction solution (50 ul) composed of 20 mM TrisHCl, pH 8.0, 50 mM NH4OAc, 5 mM DTT, 0.005% Brij-35, 5 % glycerol, 2 ug/ml kDNA, 15 uM ATP and 1 nM E. coli topoisomerase IV was incubated at room temperature for 25 min. The reaction is stopped by addition of 5 ul of 0.4 M EDTA. 50 ul of the solution was passed through a 0.2 um pore-size filer. The filter was washed with 50 ul of a buffer composed of 10 mM TrisHCl, 10 mM NaCl, pH 7.5, three times. The filtrate was collected and the decatenated DNA in the filtrate was detected by addition of 50 ul of SYBR Green II solution and measurement of the fluorescence signal at a 535 nm wavelength with an excitation wavelength of 485 nm. For compound inhibition, in the DNA decatenation reaction solution, 2 ul of the compound was included. By using a range of compound concentration, the IC50 was calculated.

Claims

102997We claim:

1. A method to determine the effect of a test compound on a decantenation enzyme, comprising: contacting a concatenated DNA, a decantenation enzyme and a test compound; separating the concatenated DNA from the decatenated DNA using a filter; and detecting the presence of filtered decatenated DNA wherein the reduction or absence of decatenated DNA is indicative that the test compound inhibits the activity of the decantenation enzyme.

2. The method of claim 1, wherein the decantenation enzyme is a topoisomerase.

3. The method of claim 2, wherein the topoisomerase is a prokaryotic topoisomerase IV or a eukaryotic topoisomerase II .

4. The method of claim 1, wherein the presence of decatenated DNA can be detected using a fluorescent dye.

5. The method of claim 4, wherein the fluorescent dye is selected from SYBR Green II, RiboGreen, and YO-PRO-I.

6. The method of claim 1, wherein the filter is a micro filter.