Note: Descriptions are shown in the official language in which they were submitted.
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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
tvpoisomerase 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 (I991),
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
supercoiIed 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
topnisomerases", 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
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various DNA topologies, but it provides inconsistent results. (See Foglesong,
P. D.;
"Fluoremetric methods employing Iow 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.: "PicoCrreen~ dsDNA (luantitation
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
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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);
(fj 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 components) 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 lOZ to about 105.
The
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concentration of the test compound in the fluid of step (a) is preferably from
about 0.1
p,g/ml to about 1000 pg/ml, more preferably from about 1 p.g/ml to about 100
p,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. 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 ~g/ml to about SO
~,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 ~tg/ml to about 10 p,g/ml, more preferably from about 0.01
pg/ml to
about I pg/ml. The fluid of step (a) preferably has a mole ratio of
DNAaopoisomerase
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°C to about SS°C, more preferably from about 35°C to
about 40°C.
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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
aI. 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 p,M,
more preferably from about 0.1 pM to about 1 ~.M. 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 leM in sterile double-distilled water. Using a standard
micropipette, 10
p,l 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 p.l of sterile double-
distilled
water in place of test compound). Next, 10 ~,1 of a master mix containing 6.0
~I of SX
incubation buffer (35mM Tris HCl (pH 7.5), 24 mM KCI, 4 mM MgCl2, 2 mM
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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 pg of relaxed DNA (Lucent Limited, UK) is
placed into each well. Then, 10 p,l of diluted gyrase (Lucent Limited, UK) in
SX storage
buffer (50 mM Tris HCl (pH 7.5) 100 mM KCI, 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°C incubator for 3 hours.
After incubation, 70 pl of a 1:400 dilution (TE buffer - 100 M Tris (pH 8.0)
and
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-DNAI - 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 1 - F(TC) - F(R-DNA)
F(S-DNA) - F(R-DNA)
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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.
