AU3269999A - Fluorescent assay for topoisomerase inhibitors - Google Patents

Description

WO 99/49077 PCT/IB99/00473 1 Fluorescent Assay for Topoisomerase Inhibitors TECHNICAL FIELD The subject invention relates to analytical procedures which use fluorescent dyes for detection of and/or for providing a quantitative measure of substances which inhibit topoisomerase enzymes, especially those of bacteria. BACKGROUND OF THE INVENTION Gyrase, a type II topoisomerase, is an essential bacterial enzyme and inhibition of gyrase by fluoroquinolones, popular commercial antibacterials, disrupts bacterial replication leading to rapid cell death. Among other functions, gyrase mediates bacterial DNA supercoiling which is essential for DNA metabolism and bacterial survival and replication. (See Wang, J. C; "DNA Topoisomerases", Annu. Rev. Biochem., vol. 65 (1996), pp. 635-692.) Limited success has been achieved in using the activity of topoisomerases on DNA as bases for topoisomerase assays. (See Andrea, J. E.; Adachi, K.; and Morgan, A. R.; "Fluorometric assays for DNA topoisomerases and topoisomerase-targeted drugs: quantitation of catalytic activity and DNA cleavage", Molec. Pharmacol., vol. 40 (1991), pp. 495-501; Lerner, C. G.; Saiki, A. Y. C.; Mackinnon, A. C., and Xuei, X.; "High throughput screen for inhibitors of bacterial DNA topoisomerase I using the scintillation proximity assay", J Biomolec. Screening, vol. 1(3) (1996), pp. 119-127; and Sandhu, L. C.; Warters, R. L.; and Dethlefsen, L. A.; "Fluorescence studies of Hoechst 33342 with supercoiled and relaxed plasmid pBR322 DNA", Cytometry, vol. 6 (1985), pp. 191 194.) Although gel-based methods to discriminate relaxed and supercoiled DNA are reported in the literature, there is a paucity of information concerning gyrase assays having potential use as automated or high throughput screening assays. (See Barrett, J. F.; Sutcliffe, J. A.; and Gootz, T. D.; "In vitro assays used to measure the activity of topoisomerases", Antimicrob. Agents Chemother., vol. 34(1) (1990), pp. 1-7.) It is known that fluorescent dyes such as ethidium bromide tend to bind differentially to WO 99/49077 PCT/IB99/00473 2 various DNA topologies, but it provides inconsistent results. (See Foglesong, P. D.; "Fluoremetric methods employing low concentrations of ethidium bromide for DNA topoisomerase and endonuclease assays", Anal. Biochem., vol. 182 (1989), pp. 284-288; and Morgan, A. R.; Lee, J. S.; Pulleyblank, D. E.; Murray, N. L.; and Evans, D. H.; "Review: ethidium fluorescence assays. Part 1. Physicochemical studies", Nucleic Acids Research, vol. 7(3) (1979), pp. 547-569.) SUMMARY OF THE INVENTION The subject invention involves methods for determining the activity of test compounds as topoisomerase inhibitors by incubating a DNA with the topoisomerase of interest, both in the presence and absence of the test compound of interest, incorporating a cyanine nucleic acid dye, and comparing the fluorescence from the dye when the test compound is present and absent. DETAILED DESCRIPTION OF THE INVENTION Commercial substrate (relaxed DNA) enzyme (gyrase) and buffers are available for performing gyrase-mediated supercoiling of DNA. The identification and application of cyanine nucleic acid dyes, such as PicoGreen® (available commercially from Molecular Probes, Inc., Eugene, OR), as nucleic acid stains for differentiating DNA topoisomers (e.g., relaxed and supercoiled DNA) represents a key discovery of the subject invention. PicoGreen® is commonly used to quantitate duplex DNA, but has not been described as a tool to effectively differentiate DNA topoisomers. (See Singer, V. L.; Jones, L. J.; Yue, S. T.; and Haugland, R. P.; "Characterization of PicoGreen reagent and development of a fluorescence-based solution assay for double-stranded DNA quantitation", Analytical Biochem. vol. 249 (1997), pp. 228-238; and Product Information Sheet of Molecular Probes, Inc.: "PicoGreen® dsDNA Quantitation Reagent and Kit", 1996.) Since the subject invention assay allows discrimination between topological isomers of duplex DNA, inhibitors (e.g., fluoroquinolones) of topoisomerases (e.g., gyrase) which catalyze the interconversion of DNA topoisomers can be identified by WO 99/49077 PCT/IB99/00473 3 drug-induced changes in DNA topology (e.g., reduction of supercoiling). Dyes such as PicoGreen® differentially bind to relaxed and supercoiled topological isomers of duplex DNA. Unlike other nucleic acid dyes, they reproducibly fluoresce more intensely upon binding to supercoiled DNA (product) following gyrase-mediated supercoiling of relaxed DNA, allowing greater discrimination between DNA topoisomers than achieved by previous assays. Inhibitors of gyrase are identifiable by reduction in fluorescence compared to control. The subject invention involves methods for determining activity of test compounds as topoisomerase inhibitors comprising the following steps: (a) mixing together, in a fluid, the test compound, a DNA, and a topoisomerase; (b) incubating the fluid from step (a); (c) incorporating a cyanine nucleic acid dye in the incubated fluid from step (b); (d) measuring the fluorescence from the dye in the fluid from step (c); (e) repeating steps (a) to (d) omitting the test compound from the fluid of step (a); (f) optionally repeating steps (a) to (d) omitting the test compound and the topoisomerase from the fluid of step (a); and (g) comparing the fluorescence measured in steps (d) and (e) or (d), (e) and (f). In step (a), the liquid component(s) of the fluid are selected such that the test compound, DNA and topoisomerase are intimately mixed, preferably as a fine suspension, or more preferably in solution. The fluid is preferably aqueous-based, but may be based on another solvent or a solvent mixture, e.g., a water/cosolvent mixture. Cosolvents mixed with water for this purpose include, e.g., dimethyl sulfoxide (DMSO) and N, N-dimethylformamide (DMF). The fluid is preferably an aqueous solution or an aqueous/cosolvent solution containing up to about 2% cosolvent. Typically, more than one concentration of the test compound is tested using a subject invention method, to ascertain the range of concentrations, and particularly the lowest level of concentration, of the test compound which might inhibit the topoisomerase enzyme of interest. Preferably from about three to about eight or more different concentrations of the test compound are tested, the ratio of highest concentration to the lowest concentration being from about 102 to about 10 5 . The WO 99/49077 PCT/IB99/00473 4 concentration of the test compound in the fluid of step (a) is preferably from about 0.1 pg/ml to about 1000 jig/ml, more preferably from about 1 pg/ml to about 100 ig/ml. The DNA is selected to complement the topoisomerase, in that a DNA that will be changed by interaction with the topoisomerase is needed. For example, gyrase (a type II topoisomerase) facilitates supercoiling of DNA. For interaction with gyrase, a relaxed DNA is preferred. A suitable, commercially-available relaxed DNA is Relaxed Plasmid pBR 322, available from Lucent Ltd., Leicester, U.K. The concentration of the DNA in the fluid of step (a) is preferably from about 0.1 pg/ml to about 100 pg/ml, more preferably from about 1 pg/ml to about 50 pg/ml. The mole ratio of test compound:DNA in the fluid of step (a) is preferably from about 1:10 -7 to about 1:10 -3 , more preferably from about 1:10-6 to about 1:10 -4 . The purpose for employing the subject invention methods is generally to identify compounds which will inhibit the topoisomerase of certain organisms of interest, particularly pathogenic bacteria. Compounds which inhibit bacterial topoisomerase may be lethal to such bacteria. Type I and type II topoisomerases are known, and are useful in the subject invention methods. Gyrase is a preferred type II topoisomerase known to facilitate supercoiling of DNA. Commercially available topoisomerases preferred for use in the subject invention methods include the following: "wild-type" gyrase, such as that from E. coli available from Lucent Ltd., and "quinolone-resistance gyrase", such as A(Trp) 2

B

2 available from Lucent Ltd. The fluid of step (a) preferably comprises the topoisomerase at a concentration from about 0.001 pg/ml to about 10 pg/ml, more preferably from about 0.01 pg/ml to about 1 pg/ml. The fluid of step (a) preferably has a mole ratio of DNA:topoisomerase of from about 10:1 to about 1:100, more preferably from about 1:1 to about 1:10. In step (b) the fluid from step (a) is incubated to allow the topoisomerase to facilitate changes in the DNA if it is not inhibited from doing so by the test compound. The incubation is preferably for a period of from about 0.2 hour to about 24 hours, more preferably from about 1 hour to about 6 hours, preferably at a temperature of from about 20 0 C to about 55oC, more preferably from about 35 0 C to about 40'C.

WO 99/49077 PCT/IB99/00473 5 In step (c) a cyanine nucleic acid dye is incorporated in the incubated fluid from step (b). Preferred cyanine nucleic acid dyes useful in the subject invention assays are disclosed in U.S. Patent Nos. 5,436,134 and 5,658,751 issued to Haugland et al. and Yue et al. on July 25, 1995 and August 19, 1997, respectively, both incorporated herein by reference. PicoGreen® is a highly preferred cyanine nucleic acid dye useful in the subject invention methods. The dye is added to the incubated fluid from step (b), resulting in a concentration of the dye in the fluid of step (c) of preferably from about 0.01 tM to about 10 pM, more preferably from about 0.1 ptM to about 1 pM. The mole ratio of DNA:dye in the fluid of step (c) is preferably from about 1:1013 to about 1:1017, more preferably from about 1:1014 to about 1:1016. In step (d), the fluorescence from the dye in the fluid from step (c) is measured using known methods. The wave lengths or excitation and emission is typically unique for each dye, and is readily ascertained without undue experimentation. Preferred for the cyanine dyes useful in the subject invention method is an excitation of from about 470 nm to about 500 nm, more preferably from about 480 nm to about 490 nm, and an emission preferably from about 510 nm to about 540 nm, more preferably from about 520 nm to about 530 nm. Examples The following is a non-limiting example of the use of a subject invention assay procedure, using standard 96-well plates useful in performing high throughput screening assays. On day of assay, compounds to be tested for activity as gyrase inhibitors are prepared at 900 gM in sterile double-distilled water. Using a standard micropipette, 10 gl of dissolved test compound is placed into individual wells of a 96 well "U" shaped microtitre plate (Dynatech, #011-010-0715) with lid (Dynatech, #001-010-5550). Column 12 contains negative and positive controls (10 pl of sterile double-distilled water in place of test compound). Next, 10 p.d of a master mix containing 6.0 pl of 5X incubation buffer (35mM Tris HCI (pH 7.5), 24 mM KC1, 4 mM MgCl 2 , 2 mM WO 99/49077 PCT/IB99/00473 6 dithiothreitol, 1.8 mM Spermidine, 6.5% glycerol (w/v), 0.1 mg/ml bovine serum albumin), 1.0 pl of 30 mM ATP, 0.25 ptg of relaxed DNA (Lucent Limited, UK) is placed into each well. Then, 10 ptl of diluted gyrase (Lucent Limited, UK) in 5X storage buffer (50 mM Tris HCI (pH 7.5) 100 mM KC1, 2 mM dithiothreitol, 1 mM EDTA, 50% glycerol (w/v)) is aliquoted into all wells with the exception of the 4 "relaxed" DNA controls (Column 12, rows A-D), which receive storage buffer without gyrase. The 96 well plates are then placed into a 37 0 C incubator for 3 hours. After incubation, 70 pl of a 1:400 dilution (TE buffer - 100 M Tris (pH 8.0) and 10 M EDTA - Sigma, # 27H6652) of PicoGreen® (Molecular Probes, Inc., # P-7581) is added to all wells, and fluorescence is measured on a FLUOstar Microplate reader (BMG Inc.) using 485 nm excitation and 525 nm emission wavelengths. The following results are obtained: F(S-DNA) = fluorescence measured from wells with supercoiled DNA (to which relaxed DNA and gyrase, but no test compound, are added). F(R-DNA) = fluorescence measured from wells with relaxed DNA (to which relaxed DNA, but no gyrase or test compound, are added). F(TC) = fluorescence measured from wells with test compound (to which relaxed DNA, gyrase, and test compound are all added). The following equation is used to calculate the signal to noise ratio in the absence of test compound: % Difference (between relaxed F(S-DNA) - F(R-DNA) and supercoiled DNA) = 100 x F(R-DNA) This calculated % Difference should be at least about 30%, or the results from the experiment may not be reliable. To calculate the degree of gyrase inhibition mediated by a particular test compound, the following equation is used: % Inhibition = 100 x - F(TC) - F(R-DNA) F(S-DNA) - F(R-DNA) WO 99/49077 PCT/IB99/00473 7 For this calculation, F(S-DNA) and F(R-DNA) are each typically averages of fluorescence for about four wells. F(TC) is typically a single reading of fluorescence for each level of test compound tested. While particular embodiments of the subject invention have been described, it will be obvious to those skilled in the arts that various changes and modifications of the subject invention can be made without departing from the spirit and scope of the invention. It is intended to cover, in the appended claims, all such modifications that are within the scope of this invention.

Claims (10)

1. A method for determining activity of a test compound as a topoisomerase inhibitor comprising the following steps: (a) mixing together in a fluid the test compound, a DNA, and a topoisomerase; (b) incubating the fluid from step (a); (c) incorporating PicoGreen® dye in the incubated fluid from step (b); (d) measuring the fluorescence from the dye in the fluid from step (c); (e) repeating steps (a) to (d) omitting the test compound from the fluid of step (a); (f) optionally repeating steps (a) to (d) omitting the test compound and the topoisomerase from the fluid of step (a); and (g) comparing the fluorescence measured in steps (d) and (e) or in steps (d), (e) and (f).

2. The method of Claim 1 wherein the DNA is a relaxed DNA, and the topoisomerase induces supercoiling of such DNA in step (e).

3. The method of Claim 1 or 2 wherein, in the fluid of step (a), the concentration of the test compound is from 0.01 gLg/ml to 1000 ptg/ml, preferably from 1 g/ml to 100 pg/ml; the concentration of the DNA is from 0.1 p.g/ml to 100 pg/ml, preferably from 1 ptg/ml to 50 gig/ml; the concentration of the topoisomerase is from 0.001 tg/ml to 10 jtg/ml, preferably from 0.01 jtg/ml to 1 ig/ml; and the concentration of the dye in the fluid of step (c) is from 0.01 4M to 10 jM, preferably from 0.1 pM to 1 tM.

4. The method of any of Claims 1-3 wherein the fluid of step (a) is aqueous-based and comprises from 0% to 2% cosolvent.

5. The method of any of Claims 1-4 wherein the fluid of step (b) is incubated at a temperature of from 20 0 C to 550C, preferably from 35 0 C to 40 0 C, for a period of from 0.2 hr to 24 hr, preferably from 1 hr to 6 hr. WO 99/49077 PCT/IB99/00473 9

6. The method of any of Claims 1-5 wherein the topoisomerase is a type II topoisomerase, preferably a gyrase, more perferably a wild-type gyrase or a quinolone resistent gyrase.

7. The method of any of Claims 1-6 wherein the mole ratio of test compound:DNA in the fluid of step (a) is from 1:10-6 to 1:10 -4 , the mole ratio of DNA:topoisomerase in the fluid of step (a) is from 1:1 to 1:10, and the mole ratio of DNA:dye in the fluid of step (c) is from 1:1014 to 1:1016.

8. The method of any of Claims 1-7 wherein step (f) is used, and preferably steps (a) to (d) are repeated using at least 3 different concentrations of the test compound in the fluid of step (a), with the ratio of the highest concentration to the lowest concentration being at least 100.

9. The method of any of Claims 1-8 wherein the fluorescence is measured using from 480 nm to 490 nm, preferably 485 nm, excitation; and from 520 nm to 530 nm, preferably 525 nm, emission wavelengths.

10. The method of any of Claims 1-9 wherein the DNA is Relaxed Plasmid pBR 322, and the gyrase is A(Trp) 2 B 2 or wild-type from E. coli.