WO1999049077A1 - Fluorescent assay for topoisomerase inhibitors - Google Patents
Fluorescent assay for topoisomerase inhibitors Download PDFInfo
- Publication number
- WO1999049077A1 WO1999049077A1 PCT/IB1999/000473 IB9900473W WO9949077A1 WO 1999049077 A1 WO1999049077 A1 WO 1999049077A1 IB 9900473 W IB9900473 W IB 9900473W WO 9949077 A1 WO9949077 A1 WO 9949077A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- dna
- fluid
- topoisomerase
- test compound
- gyrase
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/533—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving isomerase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/99—Isomerases (5.)
Definitions
- 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.
- Gyrase a type II topoisomerase
- fluoroquinolones popular commercial antibacterials
- gyrase mediates bacterial DNA supercoiling which is essential for DNA metabolism and bacterial survival and replication.
- 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.
- inhibitors e.g., fluoroquinolones
- topoisomerases e.g., gyrase
- 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:
- step (c) incorporating a cyanine nucleic acid dye in the incubated fluid from step (b);
- step (e) repeating steps (a) to (d) omitting the test compound from the fluid of step (a);
- step (f) optionally repeating steps (a) to (d) omitting the test compound and the topoisomerase from the fluid of 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).
- DMSO dimethyl sulfoxide
- DMF N, N-dimethylformamide
- the fluid is preferably an aqueous solution or an aqueous/cosolvent solution containing up to about 2% cosolvent.
- 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.
- concentrations 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 10 ⁇ to about 10 ⁇ .
- concentration of the test compound in the fluid of step (a) is preferably from about 0J ⁇ g/ml to about 1000 ⁇ g/ml, more preferably from about 1 ⁇ g/ml to about 100 ⁇ g/ml.
- the DNA is selected to complement the topoisomerase, in that a DNA that will be changed by interaction with the topoisomerase is needed.
- gyrase a type II topoisomerase
- 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 0J ⁇ g/ml to about 100 ⁇ g/ml, more preferably from about 1 ⁇ g/ml to about 50 ⁇ g/ml.
- the mole ratio of test compound:DNA in the fluid of step (a) is preferably from about 1 :10" ⁇ 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)2B2 available from Lucent Ltd.
- the fluid of step (a) preferably comprises the topoisomerase at a concentration from about 0.001 ⁇ g/ml to about 10 ⁇ g/ml, more preferably from about 0.01 ⁇ g/ml to about 1 ⁇ g/ml.
- the fluid of step (a) preferably has a mole ratio of DNAJopoisomerase of from about 10:1 to about 1 : 100, more preferably from about 1 : 1 to about 1 :10.
- 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°C to about 55°C, more preferably from about 35°C to about 40°C. 5
- 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 ⁇ M to about 10 ⁇ M, more preferably from about 0.1 ⁇ M to about 1 ⁇ M.
- the mole ratio of DNA:dye in the fluid of step (c) is preferably from about 1 :10*3 0 a bout 1 :10*7, more preferably from about 1 :10 14 to about 1 :10 16 .
- 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.
- dithiothreitol 1.8 mMspermidine, 6.5% glycerol (w/v), 0J mg/ml bovine serum albumin), 1.0 ⁇ l of 30 mM ATP, 0.25 ⁇ g of relaxed DNA (Lucent Limited, UK) is placed into each well.
- 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).
- This calculated % Difference should be at least about 30%, or the results from the experiment may not be reliable.
- 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.
Abstract
Description
Claims
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
IL13853299A IL138532A0 (en) | 1998-03-25 | 1999-03-22 | Fluorescent assay for topoisomerase inhibitors |
CA002324344A CA2324344A1 (en) | 1998-03-25 | 1999-03-22 | Fluorescent assay for topoisomerase inhibitors |
EP99942591A EP1086242A1 (en) | 1998-03-25 | 1999-03-22 | Fluorescent assay for topoisomerase inhibitors |
AU32699/99A AU3269999A (en) | 1998-03-25 | 1999-03-22 | Fluorescent assay for topoisomerase inhibitors |
JP2000538035A JP2002507433A (en) | 1998-03-25 | 1999-03-22 | Fluorescence assay for topoisomerase inhibitors |
NO20004753A NO20004753L (en) | 1998-03-25 | 2000-09-22 | Fluorescent Assay for Topoisomerase Inhibitors |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US7926598P | 1998-03-25 | 1998-03-25 | |
US60/079,265 | 1998-03-25 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1999049077A1 true WO1999049077A1 (en) | 1999-09-30 |
Family
ID=22149463
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/IB1999/000473 WO1999049077A1 (en) | 1998-03-25 | 1999-03-22 | Fluorescent assay for topoisomerase inhibitors |
Country Status (7)
Country | Link |
---|---|
EP (1) | EP1086242A1 (en) |
JP (1) | JP2002507433A (en) |
AU (1) | AU3269999A (en) |
CA (1) | CA2324344A1 (en) |
IL (1) | IL138532A0 (en) |
NO (1) | NO20004753L (en) |
WO (1) | WO1999049077A1 (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001098540A2 (en) * | 2000-06-22 | 2001-12-27 | San Diego State University Foundation | Recombination modulators and methods for their production and use |
WO2006051303A1 (en) * | 2004-11-11 | 2006-05-18 | Plant Bioscience Limited | Assay for measuring an enzyme’s capability to modify supercoil topology of nucleic acids and modulators |
WO2010044101A1 (en) * | 2008-10-08 | 2010-04-22 | V. B. Medicare Pvt. Ltd. | Purification and assay for topoisomerases and use of the assay for screening modulators of topoisomerases |
WO2012109617A2 (en) * | 2011-02-10 | 2012-08-16 | University Of Central Florida Research Foundation, Inc. | Method of assaying dna topoisomerases and dna binding proteins using high throughput screening |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996021156A1 (en) * | 1995-01-04 | 1996-07-11 | Abbott Laboratories | Method for preparing scintillation proximity assay targets |
US5759768A (en) * | 1991-05-17 | 1998-06-02 | Dana Farber Cancer Institute | Assays for factors affecting circularization of DNA, assays for factors affecting DNA integration, factors, and uses thereof |
-
1999
- 1999-03-22 EP EP99942591A patent/EP1086242A1/en not_active Withdrawn
- 1999-03-22 JP JP2000538035A patent/JP2002507433A/en not_active Withdrawn
- 1999-03-22 IL IL13853299A patent/IL138532A0/en unknown
- 1999-03-22 CA CA002324344A patent/CA2324344A1/en not_active Abandoned
- 1999-03-22 WO PCT/IB1999/000473 patent/WO1999049077A1/en not_active Application Discontinuation
- 1999-03-22 AU AU32699/99A patent/AU3269999A/en not_active Abandoned
-
2000
- 2000-09-22 NO NO20004753A patent/NO20004753L/en not_active Application Discontinuation
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5759768A (en) * | 1991-05-17 | 1998-06-02 | Dana Farber Cancer Institute | Assays for factors affecting circularization of DNA, assays for factors affecting DNA integration, factors, and uses thereof |
WO1996021156A1 (en) * | 1995-01-04 | 1996-07-11 | Abbott Laboratories | Method for preparing scintillation proximity assay targets |
Non-Patent Citations (1)
Title |
---|
OSBURNE, MARCIA S. ET AL: "An assay for the detection of bacterial DNA gyrase inhibitors", J. ANTIBIOT. (1993), 46(11), 1764-6 CODEN: JANTAJ;ISSN: 0021-8820, XP002107170 * |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001098540A2 (en) * | 2000-06-22 | 2001-12-27 | San Diego State University Foundation | Recombination modulators and methods for their production and use |
WO2001098540A3 (en) * | 2000-06-22 | 2003-04-24 | Univ State San Diego | Recombination modulators and methods for their production and use |
WO2006051303A1 (en) * | 2004-11-11 | 2006-05-18 | Plant Bioscience Limited | Assay for measuring an enzyme’s capability to modify supercoil topology of nucleic acids and modulators |
US7838230B2 (en) | 2004-11-11 | 2010-11-23 | Plant Bioscience Limited | Assay for measuring an enzyme's capability to modify supercoil topology of nucleic acids and modulators |
WO2010044101A1 (en) * | 2008-10-08 | 2010-04-22 | V. B. Medicare Pvt. Ltd. | Purification and assay for topoisomerases and use of the assay for screening modulators of topoisomerases |
WO2012109617A2 (en) * | 2011-02-10 | 2012-08-16 | University Of Central Florida Research Foundation, Inc. | Method of assaying dna topoisomerases and dna binding proteins using high throughput screening |
WO2012109617A3 (en) * | 2011-02-10 | 2012-12-06 | University Of Central Florida Research Foundation, Inc. | Method of assaying dna topoisomerases and dna binding proteins using high throughput screening |
Also Published As
Publication number | Publication date |
---|---|
EP1086242A1 (en) | 2001-03-28 |
AU3269999A (en) | 1999-10-18 |
CA2324344A1 (en) | 1999-09-30 |
IL138532A0 (en) | 2001-10-31 |
NO20004753D0 (en) | 2000-09-22 |
NO20004753L (en) | 2000-11-23 |
JP2002507433A (en) | 2002-03-12 |
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