U.S. patent application number 11/093399 was filed with the patent office on 2005-08-18 for beta-lactamase substrates having phenolic ethers.
This patent application is currently assigned to Howard Hughes Medical Center. Invention is credited to Rao, Jianghong, Tsien, Roger Y..
Application Number | 20050181469 11/093399 |
Document ID | / |
Family ID | 22992751 |
Filed Date | 2005-08-18 |
United States Patent
Application |
20050181469 |
Kind Code |
A1 |
Tsien, Roger Y. ; et
al. |
August 18, 2005 |
Beta-lactamase substrates having phenolic ethers
Abstract
Provided are fluorescent substrates for .beta.-lactamases having
the general formula I: 1 in which R is a benzyl, 2-thienylmethyl,
or cyanomethyl group; R' is selected from the group consisting of
H, physiologically acceptable salts or metal, ester groups,
ammonium cations, --CHR.sub.2OCO(CH.sub.2).sub.nC- H.sub.3,
--CHR.sub.2OCOC(CH.sub.3).sub.3, acylthiomethyl,
acyloxy-alpha-benzyl, deltabutyrolactonyl,
methoxycarbonyloxymethyl, phenyl, methylsulphinylmethyl,
.beta.-morpholinoethyl, dialkylaminoethyl, and
dialkylaminocarbonyloxymethyl, in which R.sub.2 is selected from
the group consisting of H and lower alkyl; A is selected from the
group consisting of S, O, SO, SO.sub.2 and CH.sub.2; and Z is a
donor fluorescent moiety. Also provided are methods of use of the
compound of general formula I.
Inventors: |
Tsien, Roger Y.; (La Jolla,
CA) ; Rao, Jianghong; (Los Angeles, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Howard Hughes Medical
Center
San Diego
CA
|
Family ID: |
22992751 |
Appl. No.: |
11/093399 |
Filed: |
March 29, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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|
11093399 |
Mar 29, 2005 |
|
|
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10044486 |
Jan 11, 2002 |
|
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60261313 |
Jan 12, 2001 |
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Current U.S.
Class: |
435/23 ; 435/32;
540/222 |
Current CPC
Class: |
C12Q 1/34 20130101; C12Q
1/6897 20130101; C07D 505/00 20130101; C07D 463/18 20130101; C07D
501/00 20130101; C07F 9/65613 20130101 |
Class at
Publication: |
435/023 ;
435/032; 540/222 |
International
Class: |
C12Q 001/37; C12Q
001/18; C07D 501/14 |
Goverment Interests
[0002] This invention was made in part with government support
under Grant No. NS27177 awarded by the National Institutes of
Health. The government may have certain rights in the invention.
Claims
What is claimed is:
1. A compound having the general formula: 12in which R is a benzyl,
2-thienylmethyl, or cyanomethyl group; R' is selected from the
group consisting of H, physiologically acceptable salts or metal,
ester groups, ammonium cations,
--CHR.sub.2OCO(CH.sub.2).sub.nCH.sub.3,
--CHR.sub.2OCOC(CH.sub.3).sub.3, acylthiomethyl,
acyloxy-alpha-benzyl, deltabutyrolactonyl,
methoxycarbonyloxymethyl, phenyl, methylsulphinylmethyl,
.beta.-morpholinoethyl, dialkylaminoethyl, and
dialkylaminocarbonyloxymethyl, in which R.sub.2 is selected from
the group consisting of H and lower alkyl; A is selected from the
group consisting of S, O, SO, SO.sub.2 and CH.sub.2; and Z is a
donor fluorescent moiety.
2. The compound of claim 1, wherein the donor fluorescent moiety is
selected from the group consisting of: 1314R.sub.3 is a linker for
the fluorescent donor.
3. The compound of claim 2, wherein the linker is selected from the
group consisting of a direct bond to a heteroatom in the
fluorescent moiety, --O(CH.sub.2).sub.n--, --S(CH.sub.2).sub.n--,
--NR.sub.2(CH.sub.2).sub.n-- -, --N.sup.+R.sub.2 (CH.sub.2).sub.n,
--OCONR.sub.2(CH.sub.2).sub.n--, --O.sub.2C(CH.sub.2).sub.n--,
--SCSNR.sub.2(CH.sub.2).sub.n--, --SCSO(CH.sub.2).sub.n--,
--S(CH.sub.2).sub.nCONR.sub.2(CH.sub.2).sub.m,
--S(CH.sub.2).sub.nNR.sub.2CO(CH.sub.2).sub.m, and 15in which
R.sub.2, n and m are as previously defined; and m is an integer
from 0 to 4.
4. The compound of claim 1, wherein the compound has the structure:
16
5. A method for detecting the presence of .beta.-lactamase activity
in a sample, comprising: contacting the sample with at least one
compound of general formula I: 17in which R is a benzyl,
2-thienylmethyl, or cyanomethyl group, or a quencher; R' is
selected from the group consisting of H, physiologically acceptable
salts or metal, ester groups, ammonium cations,
--CHR.sub.2OCO(CH.sub.2).sub.nCH.sub.3,
--CHR.sub.2OCOC(CH.sub.3).sub.3, acylthiomethyl,
acyloxy-alpha-benzyl, deltabutyrolactonyl,
methoxycarbonyloxymethyl, phenyl, methylsulphinylmethyl,
.beta.-morpholinoethyl, dialkylaminoethyl, and
dialkylaminocarbonyloxymethyl, in which R.sub.2 is selected from
the group consisting of H and lower alkyl; A is selected from the
group consisting of S, O, SO, SO.sub.2 and CH.sub.2; and Z is a
donor fluorescent moiety.
6. The method of claim 5, wherein said sample has a
.beta.-lactamase reporter gene.
7. The method of claim 6, wherein said .beta.-lactamase reporter
gene is in a mammalian cell.
8. The method of claim 5, wherein samples having .beta.-lactamase
activity are separated from samples having no .beta.-lactamase
activity by fluorescent-activated cell sorting.
9. The method of claim 5, wherein the .beta.-lactamase activity
results from a .beta.-lactamase enzyme that was prepared by
mutagenesis of another .beta.-lactamase enzyme.
10. The method of claim 5, wherein said compound is a membrane
permeant derivative.
11. The method of claim 5, wherein the donor fluorescent moiety is
selected from the group consisting of: 1819R.sub.3 is a linker for
the fluorescent donor.
12. The method of claim 11, wherein the linker is selected from the
group consisting of a direct bond to a heteroatom in the
fluorescent moiety, --O(CH.sub.2).sub.n--, --S(CH.sub.2).sub.n--,
--NR.sub.2(CH.sub.2).sub.n-- -, --N.sup.+R.sub.2(CH.sub.2).sub.n,
--OCONR.sub.2(CH.sub.2).sub.n--, --O.sub.2C(CH.sub.2).sub.n--,
--SCSNR.sub.2(CH.sub.2).sub.n--, --SCSO(CH.sub.2).sub.n--,
--S(CH.sub.2).sub.nCONR.sub.2(CH.sub.2).sub.m,
--S(CH.sub.2).sub.nNR.sub.2CO(CH.sub.2).sub.m, and 20in which
R.sub.2, n and m are as previously defined; and m is an integer
from 0 to 4.
13. The method of claim 5, wherein the compound has the structure:
21
14. A method for determining whether a compound of claim 1 is a
substrate for a .beta.-lactamase enzyme, comprising: contacting
said compound with a sample containing said .beta.-lactamase
enzyme; exciting at the wavelength for the said compound when
cleaved; and measuring fluorescence.
15. The method of claim 14, wherein said compound is a membrane
permeant derivative.
16. The method of claim 14, wherein said .beta.-lactamase enzyme
has been prepared by mutagenesis of another .beta.-lactamase
enzyme.
Description
RELATED APPLICATION DATA
[0001] This application claims priority under 35 U.S.C. .sctn. 119
to U.S. Provisional Application Ser. No. 60/261,313, filed Jan. 12,
2001 the contents of which is incorporated by reference in its
entirety herein.
FIELD OF THE INVENTION
[0003] The present invention relates generally to the fields of
chemistry and biology. More particularly, the present invention
relates to compositions and methods for use in measuring gene
expression.
BACKGROUND
[0004] A reporter gene assay measures the activity of a gene's
promoter. It takes advantage of molecular biology techniques, which
allow one to put heterologous genes under the control of any
promoter and introduce the construct into the genome of a mammalian
cell (Gorman, C. M. et al., Mol. Cell Biol. 2:1044-1051 (1982);
Alam, J. and Cook, J. L., Anal. Biochem. 188:245-254, (1990)).
Activation of the promoter induces the reporter gene as well as or
instead of the endogenous gene. By design the reporter gene codes
for a protein that can easily be detected and measured. Commonly it
is an enzyme that converts a commercially available substrate into
a product. This conversion is conveniently followed by either
chromatography or direct optical measurement and allows for the
quantification of the amount of enzyme produced.
[0005] Reporter genes are commercially available on a variety of
plasmids for the study of gene regulation in a large variety of
organisms (Alam and Cook, supra). Promoters of interest can be
inserted into multiple cloning sites provided for this purpose in
front of the reporter gene on the plasmid (Rosenthal, N., Methods
Enzymol. 152:704-720 (1987); Shiau, A. and Smith, J. M., Gene
67:295-299 (1988)). Standard techniques are used to introduce these
genes into a cell type or whole organism (e.g., as described in
Sambrook, J., Fritsch, E. F. and Maniatis, T. Expression of cloned
genes in cultured mammalian cells. In: Molecular Cloning, edited by
Nolan, C. New York: Cold Spring Harbor Laboratory Press, 1989).
Resistance markers provided on the plasmid can then be used to
select for successfully transfected cells.
[0006] Ease of use and the large signal amplification make this
technique increasingly popular in the study of gene regulation.
Every step in the cascade
DNA.fwdarw.RNA.fwdarw.Enzyme.fwdarw.Product.fwdarw.Signal amplifies
the next one in the sequence. The further down in the cascade one
measures, the more signal one obtains.
[0007] In an ideal reporter gene assay, the reporter gene under the
control of the promoter of interest is transfected into cells,
either transiently or stably. Receptor activation leads to a change
in enzyme levels via transcriptional and translational events. The
amount of enzyme present can be measured via its enzymatic action
on a substrate. The substrate is a small uncharged molecule that,
when added to the extracellular solution, can penetrate the plasma
membrane to encounter the enzyme. A charged molecule can also be
employed, but the charges need to be masked by groups that will be
cleaved by endogenous cellular enzymes (e.g., esters cleaved by
cytoplasmic esterases).
[0008] For a variety of reasons, the use of substrates which
exhibit changes in their fluorescence spectra upon interaction with
an enzyme are particularly desirable. In some assays, the
fluorogenic substrate is converted to a fluorescent product.
Alternatively, the fluorescent substrate changes fluorescence
properties upon conversion at the reporter enzyme. The product
should be very fluorescent to obtain maximal signal, and very
polar, to stay trapped inside the cell.
[0009] To achieve the highest possible sensitivity in a reporter
assay one has to maximize the amount of signal generated by a
single reporter enzyme. An optimal enzyme will convert 10.sup.5
substrate molecules per second under saturating conditions (Stryer,
L. Introduction to enzymes. In: Biochemistry, New York: W. H.
Freeman and company, 1981, pp. 103-134). .beta.-Lactamases will
cleave about 10.sup.3 molecules of ideal substrates per second
(Chang, Y. H. et al., Proc. Natl. Acad. Sci. USA 87:2823-2827
(1990)). Using a fluorogenic substrate one can obtain up to
10.sup.6 photons per fluorescent product produced, depending on the
type of dye used, when exciting with light of the appropriate
wavelength. The signal terminates with the bleaching of the
fluorophore (Tsien, R. Y. and Waggoner, A. S. Fluorophores for
confocal microscopy: Photophysics and photochemistry. In: Handbook
of Biological Confocal Microscopy, edited by Pawley, J. B. Plenum
Publishing Corporation, 1990, pp. 169-178). These numbers
illustrate the theoretical magnitude of signal obtainable in this
type of measurement. In practice a minute fraction of the photons
generated will be detected, but this holds true for fluorescence,
bioluminescence or chemiluminescence. A good fluorogenic substrate
for a reporter enzyme has to have a high turnover at the enzyme in
addition to good optical properties such as high extinction and
high fluorescence quantum yield.
SUMMARY OF THE INVENTION
[0010] The novel .beta.-lactamase substrates disclosed herein are
easily synthesized. Prior .beta.-lactamase substrates consist of a
donor fluorophore and an acceptor chromophore connected by a
cephalosporin. Fluorescence resonance energy transfer between the
donor and acceptor is disrupted by .beta.lactamase cleavage of the
cephalosporin. The novel substrates disclosed herein, are simpler
phenolic ethers of cephalosporins in which .beta.-lactamase attack
releases the free phenolic chromophore, which is then detectable by
fluorescence, chemiluminescence, or formation of colored
precipitates. One advantage over prior substrates are that the
novel molecules are smaller, can more readily give long-wavelength
absorbencies or fluorescence and give lower detection limits.
[0011] In one embodiment, the present invention provides compounds
that are substrates for .beta.-lactamase that are suitable for use
in a reporter gene assay. It is a further object of the invention
to provide membrane-permeant compounds that can be transformed into
substantially membrane-impermeant compounds after entry into a
cell.
[0012] In accordance with the present invention, compounds are
provided having general formula I: 2
[0013] in which R is a benzyl, 2-thienylmethyl, or cyanomethyl
group, or a quencher; R' is selected from the group consisting of
H, physiologically acceptable salts or metal, ester groups,
ammonium cations, --CHR.sub.2OCO(CH.sub.2).sub.nCH.sub.3,
--CHR.sub.2OCOC(CH.sub.3).sub.3, acylthiomethyl,
acyloxy-alpha-benzyl, deltabutyrolactonyl,
methoxycarbonyloxymethyl, phenyl, methylsulphinylmethyl,
.beta.-morpholinoethyl, dialkylaminoethyl, and
dialkylaminocarbonyloxymet- hyl, in which R.sub.2 is selected from
the group consisting of H and lower alkyl; A is selected from the
group consisting of S, O, SO, SO.sub.2 and CH.sub.2; and Z is a
donor fluorescent moiety.
[0014] In another aspect, the present invention provides a method
for determining whether a .beta.-lactamase enzyme can cleave a
compound of the present invention having the general formula I, or
a membrane permeant derivative thereof. The method involves
contacting a sample containing the enzyme with a compound of the
present invention, exciting the sample with radiation of one or
more wavelengths that are suitable for the cleaved compound, and
determining the degree of fluorescence emitted from the sample. A
degree of fluorescence emitted from the sample that is greater than
an expected degree indicates that the .beta.-lactamase enzyme can
cleave the compound and that the compound is a substrate for the
.beta.-lactamase enzyme.
[0015] In another aspect, the present invention provides methods
for determining whether a sample contains .beta.-lactamase
activity. The method involves contacting the sample with a compound
of the present invention having general formula I, exciting the
sample with radiation of one or more wavelengths that are suitable
for the cleaved compound, and determining the degree of
fluorescence emitted from the sample. A degree of fluorescence
emitted from the sample that is greater than an expected degree
indicates the presence of .beta.-lactamase activity in the sample.
One aspect of this method is for determining the amount of an
enzyme in a sample by determining the degree of fluorescence
emitted at a first and second time after contacting the sample with
a compound of the present invention. The difference in the degree
of fluorescence emitted from the sample at the first and second
time is determined. That difference reflects the amount of a
.beta.-lactamase enzyme in the sample.
[0016] In another aspect, the present invention is directed to
screening assays using the compounds having general formula I of
the present invention and a host cell, such as a mammalian cell,
transfected with at least one recombinant nucleic acid molecule
encoding at least one protein having .beta.-lactamase activity.
Such recombinant nucleic acid molecule comprise expression control
sequences adapted for function in a eukaryotic cell, such as a
vertebrate cell, operatively linked to a nucleotide sequence coding
for the expression of a lactamase enzyme. The present invention
also provides recombinant nucleic acid molecules comprising
expression control sequences adapted for function in a eukaryotic
cell, such as a vertebrate cell, operably linked to a nucleotide
sequence coding for the expression of a cytosolic .beta.-lactamase
enzyme.
[0017] In another aspect, the present invention provides methods
for determining the amount of .beta.-lactamase activity in a cell.
This method involves providing a sample comprising a host cell
transfected with a recombinant nucleic acid molecule having an
expression control sequences operatively linked to nucleic acid
sequences coding for the expression of a .beta.-lactamase enzyme.
The sample can comprise whole host cells, or an extract of the host
cells, which is contacted with a compound of the present invention.
The amount of compound cleaved is measured, whereby the amount of
substrate cleaved is related to the amount of .beta.-lactamase
activity in the host cell.
[0018] In another aspect, the present invention provides methods
for monitoring the expression of a gene operably linked to a set of
expression control sequences. The methods involve providing a host
eukaryotic cell transfected with a recombinant nucleic acid
molecule. The nucleic acid molecule comprises an expression control
sequence operatively linked to nucleic acid sequences coding for
the expression of a .beta.-lactamase enzyme. If the host eukaryotic
cell is a fungus, the .beta.-lactamase is a cytosolic
.beta.-lactamase enzyme. A sample comprising the host eukaryotic
cell, or an extract or conditioned medium produced therefrom or
thereby, is contacted with a compound of the present invention. The
amount of compound cleaved is determined using the methods of the
present invention, wherein the amount of substrate cleaved is
related to the amount of .beta.-lactamase activity in the host
eukaryotic cell, which is related to the expression of the
gene.
[0019] In another aspect, the present invention provides methods
for determining whether a test compound alters the expression of a
gene operably linked to a set of expression control sequences. The
methods involve providing a host eukaryotic cell transfected with a
recombinant nucleic acid construct. The recombinant nucleic acid
construct comprises a set of expression control sequences operably
linked to nucleic acid sequences coding for the expression of a
.beta.-lactamase enzyme. The host eukaryotic cell is contacted with
the test compound. This host eukaryotic cell is then contacted with
a compound of the present invention. The amount of the compound of
the present invention cleaved is then measured using the methods of
the present invention, whereby the amount of the compound of the
present invention cleaved is related to the amount of
.beta.-lactamase activity in the cell.
[0020] In another aspect, the present invention provides methods of
clonal selection by providing cells transfected with a recombinant
nucleic acid molecule comprising at least one expression control
sequences operably linked to at least one nucleic acid sequence
coding for the expression of a cytosolic .beta.-lactamase enzyme.
The cells are contacted with a substance that activates, inhibits,
or has no effect on the activation of the expression control
sequence. The cells are contacted with a compound of the present
invention. The amount of the compound of the present invention
cleaved is determined within individual cells (including each
individual cell), whereby the amount of the compound of the present
invention cleaved reflects the amount of .beta.-lactamase activity
in the cells. Cells having a selected level of .beta.-lactamase
activity are selected and propagated.
[0021] Another aspect of the present invention is to use a
.beta.-lactamase reporter gene and a compound of the present
invention to screen test chemicals for biochemical. The method
includes providing cells transfected with a recombinant nucleic
acid molecule. The recombinant nucleic acid molecule comprises at
least one expression control sequence operably linked to at least
one nucleic acid sequence encoding for the expression of a
.beta.-lactamase enzyme. The cells are contacted with a test
chemical that may activate, inhibit, or have no effect on the
activation of the expression control sequence. The cells are
contacted with a compound of the present invention and the amount
of the compound cleaved is measured. The amount of compound cleaved
reflects the amount of .beta.-lactamase activity within the at
least one cell, which reflects a biochemical activity within the at
least one cell.
BRIEF DESCRIPTION OF FIGURES
[0022] FIG. 1 shows the new substrate is synthetically easily
accessible.
[0023] FIG. 2 shows enzymatic fragmentation can take place to the
new substrate.
[0024] FIG. 3 shows synthesis of RECTO.
[0025] FIG. 4 shows oxidation state of the sulfide affects
stability of the substrate.
[0026] FIG. 5 shows sulfoxide increases substrate stability.
[0027] FIG. 6 shows increased resorufin deposition in
.beta.-lactamase-transfected vs. wild type cells.
[0028] FIG. 7 shows cephalosporin-phenol.
[0029] FIG. 8 shows resorufin-cephalosporin cleaved by
.beta.-lactamase.
[0030] FIG. 9 shows absorption spectra of resorufin-cephalosporin
before and after .beta.-lactamase treatment.
[0031] FIG. 10 shows fluorescence emission of
resorufin-cephalosporin before and after .beta.-lactamase
treatment.
DETAILED DESCRIPTION OF THE INVENTION
[0032] Definitions
[0033] In accordance with the present invention and as used herein,
the following terms are defined with the following meanings, unless
stated otherwise.
[0034] The term "fluorescent donor moiety" refers the radical of a
fluorogenic compound which can absorb energy and is capable of
transferring the energy to another fluorogenic molecule or part of
a compound. Suitable donor fluorogenic molecules include, but are
not limited to, coumarins and related dyes; xanthene dyes such as
fluoresceins, rhodols, and rhodamines; resorufins; cyanine dyes;
bimanes; acridines; isoindoles; dansyl dyes; aminophthalic
hydrazides such as luminol and isoluminol derivatives;
aminophthalimides; aminonaphthalimides; aminobenzofurans;
aminoquinolines; dicyanohydroquinones; and europium and terbium
complexes and related compounds. Accordingly, a donor fluorescent
moiety can be a dye or chromophore.
[0035] The term "quencher" refers to a chromophoric molecule or
part of a compound which is capable of reducing the emission from a
fluorescent donor when attached to the donor. Quenching may occur
by any of several mechanisms including, for example, fluorescence
resonance energy transfer, photoinduced electron transfer,
paramagnetic enhancement of intersystem crossing, Dexter exchange
coupling, and exciton coupling such as the formation of dark
complexes. The term "acceptor" as used herein refers to a quencher
which operates via fluorescence resonance energy transfer. Many
acceptors can reemit the transferred energy as fluorescence.
Examples include coumarins and related fluorophores, xanthenes such
as fluoresceins, rhodols and rhodamines, resorufins, cyanines,
difluoroboradiazaindacenes, and phthalocyanines. Other chemical
classes of acceptors generally do not re-emit the transferred
energy. Examples include indigos, benzoquinones, anthraquinones,
azo compounds, nitro compounds, indoanilines, di- and
triphenylmethanes.
[0036] The term "dye" refers to a molecule or part of a compound
which absorbs specific frequencies of light, including, but not
limited to, ultraviolet light. The terms "dye" and "chromophore"
are used herein synonymously.
[0037] The term "fluorophore" refers to chromophore or dye which
fluoresces.
[0038] The term "membrane-permeant derivative" means a chemical
derivative of a compound of general formula I containing at least
one acylated aromatic hydroxyl, acylated amine, or alkylated
aromatic. hydroxyl wherein the acyl group contains 1 to 5 carbon
atoms and wherein the alkyl group is selected from the group
consisting of --CH.sub.2OC(O)alk, --CH.sub.2SC(O)alk,
--CH.sub.2OC(O)Oalk, lower acyloxy-alpha-benzyl, and
deltabutyrolactonyl; wherein alk is lower alkyl of 1 to 4 carbon
atoms. These derivatives are better able to cross cell membranes,
i.e. membrane permeant, because hydrophilic groups are masked to
provide more hydrophobic derivatives. Also, the masking groups are
designed to be cleaved from the fluorogenic substrate within the
cell to generate the derived substrate intracellularly. Because the
substrate is more hydrophilic than the membrane permeant derivative
it is now trapped within the cells.
[0039] The term "alkyl" refers to straight, branched, and cyclic
aliphatic groups of 1 to 8 carbon atoms, preferably 1 to 6 carbon
atoms, and most preferably 1 to 4 carbon atoms. The term "lower
alkyl" refers to straight and branched chain alkyl groups of 1 to 4
carbon atoms.
[0040] The term "aliphatic" refers to saturated and unsaturated
alkyl groups of 1 to 10 carbon atoms, preferably 1 to 6 carbon
atoms, and most preferably 1 to 4 carbon atoms.
[0041] The term ".beta.-lactamase" refers to an enzyme that can
cleave a .beta.-lactam ring. Examples of a .beta.-lactamase enzyme
include the naturally occurring forms of .beta.-lactamase and
enzymes that have been prepared by mutagenesis of .beta.-lactamase
enzymes. If a .beta.-lactamase enzyme can cleave the .beta.-lactam
ring in particular compound having the general formula I (or its
membrane permeant derivative), then this particular compound is a
substrate of this invention for this particular .beta.-lactamase
enzyme (see, for example, WO 96/30540, published Oct. 3, 1996).
[0042] .beta.-Lactamases are excellent enzymes due to their
diffusion-controlled catalysis of .beta.-lactam hydrolysis
(Christensen, H. et al., Biochem. J. 266:853-861 (1990)). Upon
examination of the other properties of this class of enzymes, it
was determined that they were suited to the task of an
intracellular reporter enzyme. They cleave the .beta.-lactam ring
of .beta.-lactam antibiotics, such as penicillins and
cephalosporins, generating new charged moieties in the process
(O'Callaghan, C. H. et al., Antimicrob. Agents. Chemother. 8:57-63,
(1968); Stratton, C. W., J. Antimicrob. Chemother. 22, Suppl. A:
23-35 (1988)). A first generation cephalosporin is illustrated
below, 1, with the arrow pointing to the site of cleavage by
.beta.-lactamase. The free amino group thus generated 2 donates
electron density through the vinyl group to promote irreversible
cleavage of a nucleofugal group R.sub.2 from the 3'-position.
R.sub.2 is thus free to diffuse away from the R.sub.1-cephalosporin
conjugate 3. 3
[0043] .beta.-Lactamases are a class of enzymes that have been very
well characterized due to their clinical relevance in making
bacteria resistant to .beta.-lactam antibiotics (Waley, S. G., Sci.
Prog. 72:579-597 (1988); Richmond, M. H. et al., Ann. N.Y. Acad.
Sci. 182:243-257 (1971)). Most .beta.-lactamases have been cloned
and their amino acid sequence determined (see, e.g., Ambler, R. P.,
Phil. Trans. R. Soc. Lond. Ser. B. 289:321-331 (1980)).
[0044] A gene encoding .beta.-lactamase is known to molecular
biologists as the ampicillin resistance gene (Amp.sup.r) and is
commonly used to select for successfully transduced bacteria
(Castagnoli, L. et al., Genet. Res. 40: 217-231 (1982)); clones
thereof are almost universally available. The enzyme catalyzes the
hydrolysis of a .beta.-lactam ring and will not accept peptides or
protein substrates (Pratt, R. F. and Govardhan, C. P., Proc. Natl.
Acad. Sci. USA 81:1302-1306 (1984); Murphy, B. P. and Pratt, R. F.,
Biochemistry 30:3640-3649 (1991)). The kinetics of this reaction is
well understood and there is no product inhibition (Bush, K. and
Sykes, R. B., Antimicrob. Agents. Chemother. 30:6-10 (1986);
Christensen et al. (1990), supra). The enzyme substrates are less
polar than the products.
[0045] The carboxyl group in the substrate can be easily masked by
an acetoxymethyl ester (Jansen, A. B. A. and Russell, T. J., J.
Chem. Soc. 2127-2132, (1965); Daehne, W. et al., J. Med. Chem.
13:607-612 (1970)), which is readily cleaved by endogenous
mammalian intracellular esterases. Conversion by these esterases
followed by cleavage of the .beta.-lactam by .beta.-lactamase
generates two negative charges and a tertiary amine. Multiple
chromogenic substrates of different design have been reported and
are commercially available (Jones, R. N. et al., J. Clin.
Microbiol. 15:677-683 (1982); Jones, R. N. et al., J. Clin.
Microbiol. 15:954-958 (1982); O'Callaghan, C. H. et al.,
Antimicrob. Agents. Chemother. 1:283-288 (1972)).
[0046] A large number of .beta.-lactamases have been isolated and
characterized, all of which would be suitable for use in accordance
with the present invention. Initially, .beta.-lactamases were
divided into different classes (I through V) on the basis of their
substrate and inhibitor profiles and their molecular weight
(Richmond, M. H. and Sykes, R. B., Adv. Microb. Physiol. 9: 31-88
(1973)). More recently, a classification system based on amino acid
and nucleotide sequence has been introduced (Ambler, R. P., Phil.
Trans. R. Soc. Lond. Ser. B. 289:321-331 (1980)). Class A
.beta.-lactamases possess a serine in the active site and have an
approximate weight of 29 kd. This class contains the
plasmid-mediated TEM .beta.-lactamases such as the RTEM enzyme of
pBR322. Class B .beta.-lactamases have an active-site zinc bound to
a cysteine residue. Class C enzymes have an active site serine and
a molecular weight of approximately 39 kd, but have no amino acid
homology to the class A enzymes.
[0047] The coding region of an exemplary .beta.-lactamase which may
be employed in the present invention is described in U.S. Pat. No.
5,955,604. The pTG2dell containing this sequence has been described
(Kadonaga, J. T. et al., J. Biol. Chem. 259:2149-2154 (1984)). The
entire coding sequence of wild-type pBR322 .beta.-lactamase has
also been published (Sutcliffe, J. G., Proc. Natl. Acad. Sci. USA
75:3737-3741 (1978)). As would be readily apparent to those skilled
in the field, this and other comparable sequences for peptides
having .beta.-lactamase activity would be equally suitable for use
in accordance with the present invention. The .beta.-lactamase
reporter gene is employed in an assay system in a manner well known
per se for the use of reporter genes (for example, in the form of a
suitable plasmid vector).
[0048] In conjunction with a suitable .beta.-lactamase, there are
employed in accordance with the present invention fluorogenic
substrates of the general formula I: 4
[0049] in which R is a benzyl, 2-thienylmethyl, or cyanomethyl
group; R' is selected from the group consisting of H,
physiologically acceptable salts or metal, ester groups, ammonium
cations, --CHR.sub.2OCO(CH.sub.2).- sub.nCH.sub.3,
--CHR.sub.2OCOC(CH.sub.3).sub.3, acylthiomethyl,
acyloxy-alpha-benzyl, deltabutyrolactonyl,
methoxycarbonyloxymethyl, phenyl, methylsulphinylmethyl,
.beta.-morpholinoethyl, dialkylaminoethyl, and
dialkylaminocarbonyloxymethyl, in which R.sub.2 is selected from
the group consisting of H and lower alkyl; A is selected from the
group consisting of S, O, SO, SO.sub.2 and CH.sub.2; and Z is a
donor fluorescent moiety, selected from the group consisting of:
56
[0050] R.sub.3 is a linker for the fluorescent donor. The linker
R.sub.3 serves the purpose of attaching the fluorescent donor to
the cephalosporin phenol ether derived backbone. Suitable linkers
for use as R.sub.3 include, but are not limited to, a direct bond
to a heteroatom (e.g., O, N or S) in the dye's chromophore or the
following: --O(CH.sub.2).sub.n--, --S(CH.sub.2).sub.n--,
--NR.sub.2(CH.sub.2).sub.n-- -, --N.sup.+R.sub.2(CH.sub.2).sub.n,
--OCONR.sub.2(CH.sub.2).sub.n--, --O.sub.2C(CH.sub.2).sub.n--,
--SCSNR.sub.2(CH.sub.2).sub.n--, --SCSO(CH.sub.2).sub.n--,
--S(CH.sub.2).sub.nCONR.sub.2(CH.sub.2).sub.m,
--S(CH.sub.2).sub.nNR.sub.2CO(CH.sub.2).sub.m, and 7
[0051] in which R.sub.2, n and m are as previously defined; and m
is an integer from 0 to 4. Particularly preferred groups are
--S(CH.sub.2).sub.n. Also preferred is H. In a one aspect, the
compounds of the present invention are membrane-permeant.
[0052] As would readily be appreciated by those skilled in the art,
the efficiency of fluorescence resonance energy transfer depends on
the fluorescence quantum yield of the donor fluorophore, the
donor-acceptor distance and the overlap integral of donor
fluorescence emission and acceptor absorption. The energy transfer
is most efficient when a donor fluorophore with high fluorescence
quantum yield (preferably, one approaching 100%) is paired with an
acceptor with a large extinction coefficient at wavelengths
coinciding with the emission of the donor. The dependence of
fluorescence energy transfer on the above parameters has been
reported (Forster, T. (1948) Ann. Physik 2:55-75; Lakowicz, J. R.,
Principles of Fluorescence Spectroscopy, New York:Plenum Press
(1983); Herman, B., Resonance energy transfer microscopy, in:
Fluorescence Microscopy of Living Cells in Culture, Part B, Methods
in Cell Biology, Vol 30, ed. Taylor, D. L. & Wang, Y.-L., San
Diego: Academic Press (1989), pp. 219-243; Turro, N.J., Modern
Molecular Photochemistry, Menlo Part: Benjamin/Cummings Publishing
Co., Inc. (1978), pp. 296-361), and tables of spectral overlap
integrals are readily available to those working in the field (for
example, Berlman, I. B. Energy transfer parameters of aromatic
compounds, Academic Press, New York and London (1973)). The
distance between donor fluorophore and acceptor dye at which
fluorescence resonance energy transfer (FRET) occurs with 50%
efficiency is termed R.sub.0 and can be calculated from the
spectral overlap integrals. For the donor-acceptor pair fluorescein
tetramethyl rhodamine which is frequently used for distance
measurement in proteins, this distance R.sub.0 is around
50-70.DELTA. (dos Remedios, C. G. et al. (1987) J. Muscle Research
and Cell Motility 8:97-117). The distance at which the energy
transfer in this pair exceeds 90% is about 45.DELTA.. When attached
to the cephalosporin backbone the distances between donors and
acceptors are in the range of 10 .DELTA. to 20 .DELTA., depending
on the linkers used and the size of the chromophores. For a
distance of 20 .DELTA., a chromophore pair will have to have a
calculated R.sub.0 of larger than 30 .DELTA. for 90% of the donors
to transfer their energy to the acceptor, resulting in better than
90% quenching of the donor fluorescence. Cleavage of such a
cephalosporin by .beta.-lactamase relieves quenching and produces
an increase in donor fluorescence efficiency in excess of tenfold.
Accordingly, it is apparent that identification of appropriate
donor-acceptor pairs for use as taught herein in accordance with
the present invention would be essentially routine to one skilled
in the art.
[0053] To measure .beta.-lactamase activity in the cytoplasm of
living cells, smaller molecular weight chromophores as hereinafter
described are in general preferred over larger ones as substrate
delivery becomes a problem for larger compounds. Large molecules,
especially those over about 1200 daltons, also tend to bind more
avidly to cellular constituents than small ones, thereby removing
at least some of them from access and cleavage by
.beta.-lactamase.
[0054] Suitable chromaphores are disclosed in U.S. Pat. No.
5,955,604, the disclosure of which is incorporated herein by
reference in its entirety.
[0055] A general method for synthesis of compounds of general
formula I is depicted below (Scheme 1). As one of ordinary skill in
the art will appreciate, the methods below can be used for a
variety of derivatives, and other methods of synthesis are
possible. 8
[0056] Table 1 depicts other cephempropenyl phenol ethers
synthesized.
1TABLE 1 Compound R A R' cis/trans Z 1 CH.sub.3 S H mix 9 2
CH.sub.3 S CH.sub.2OAc mix same as above 3 10 S H mix same as above
4 same S CH.sub.2OAc mix same as above 5 same SO H cis same as
above 6 same SO H trans same as above 7 same SO CH.sub.2OAc mix
same as above 8 same SO.sub.2 H mix same as above 9 same SO.sub.2
CH.sub.2OAc mix same as above 10 same SO CHPh.sub.2 mix 11
[0057] The cephalosporin starting materials are commercially
available cephalosporin derivatives 7-aminocephalosporanic acid or
7-amino 3'-chlorocephalosporanic acid as its benzhydryl or tertiary
butyl ester (R.sub.0).
[0058] A large variety of phenolic fluorophores could be
substituted for the resorufin derivative disclosed herein. Examples
include the courmarin, the pyrene, and the rhodol. In each case the
fluorescence is greatly enhanced and shifts to longer wavelengths
when the free phenolic group is release from the ether linkage to
the cephalosporin.
[0059] Another variety of fluorophore formation is exemplified by
the fluorosalicylate ether. Once the free fluorosalicylate is
released it forms a mixed chelate with terbium-EDTA or
europium-EDTA, which would be provided as an additional component
of the assay system. Excitation of the fluorosalicylate causes
energy transfer to the lanthanide ion, which then emits with
extremely sharp peaks and millisecond-long fluorescence lifetimes.
Both the latter properties make this fluorescence very distinctive
and easy to separate from autofluorescence backgrounds.
[0060] A chemiluminescence readout can also be generated by use of
the adamantylidene-dioxetane. The release of the free phenol
triggers spontaneous fragmentation of the dioxetane and emission of
light. Another version is the luciferin ether. In this case ATP is
added and luciferase to get the light output. Only free luciferin,
not a luciferin derivative is a substrate for the enzyme. The
advantage over the adamanylidene-dioxetane would be the much higher
quantum efficiency of the luciferase-catalyzed chemiluminescence
compared to the non-enzymatic glow.
[0061] Colored or fluorescent precipitates should result from the
indolyl or 2-(2-hydroxyphenyl)quinazolin-4-one substrates. Release
of the free phenol triggers oxidation of 3-hydroxyindoles to blue
indigo precipitates. The free 2-(2-hydroxyphenyl)quinazolin-4-one
likewise forms a brightly fluorescent precipitate.
[0062] It is also possible to couple two cephalosporins to a
bis(phenol) such as the fluorescein. Only when both phenols are
freed does the fluorescein become fully fluorescent.
[0063] The cephalosporin backbone serves as a cleavable linker.
After cleavage it provides the charges necessary to keep a dye
inside the cell. Dyes may be chosen in a manner that one dye
absorbs light (quencher or acceptor chromophore) at the wavelength
that the other one emits (donor fluorophore). In the intact
cephalosporin the two dyes are in close proximity to each other.
When exciting the donor fluorophore one observes fluorescence
resonance energy transfer (FRET) from the donor to the acceptor
instead of donor fluorescence (Forster, T., Ann. Physik 2: 55-75
(1948)). If the acceptor is a nonfluorescent dye the energy is
given off to the solvent; the donor fluorescence is quenched. In
the case of the acceptor being itself a fluorescent dye,
fluorescence re-emission occurs at the acceptor's emission
wavelength. In polar solvents such as water, hydrophobic donor and
acceptor fluorophores can stack when separated by a short flexible
linker. Due to this association in the ground state, a dark complex
is formed (Yaron, A. et al., Anal. Biochem. 95: 228-235 (1979)). In
this complex, neither fluorophore can emit light, causing the
fluorescence of both dyes to be quenched (Bojarski, C. and
Sienicki, K. Energy transfer and migration in fluorescent
solutions. In: Photochemistzy and Photophysics, edited by Rabek, J.
F. Boca Raton: CRC Press, Inc., 1990, pp. 1-57). In either case, a
large change in fluorescence goes along with .beta.-lactam
cleavage, which can be used to measure .beta.-lactamase activity.
As both dyes diffuse away from each other, stacking and energy
transfer are disrupted. Cephalosporins carrying a donor and an
acceptor dye which fluoresces are referred to herein as
FRET-cephalosporins.
[0064] Fluorescence resonance energy transfer has been used as a
spectroscopic ruler for measuring molecular distances in proteins
and peptides as it is effective in the range from 10-100 angstroms.
This energy transfer is proportional to the inverse sixth power of
the distance between donor and acceptor. Its efficiency is higher,
the better donor emission and acceptor absorbance overlap, and the
longer the fluorescence lifetime of the donor (in absence of the
acceptor). FRET can be very efficient over distances of 10-20
angstroms.
[0065] In the cephalosporin, distances for attachment of donor and
acceptor are greater than 10 angstroms and a minimum of 10
bond-lengths, if one includes the two minimal spacers at 7- and
3-positions. Over this distance FRET is very efficient, if the
right donor-acceptor pairs are chosen. Upon cleavage, fluorescence
increases due to loss of the quencher dye.
[0066] The fluorogenic substrates of the invention are initially
colorless and nonfluorescent outside cells. The substrates are
designed so they readily cross cell membranes into the cytoplasm,
where they are converted to fluorescent compounds by endogenous
nonspecific esterases and stay trapped due to their charges. In the
intact molecules, fluorescence energy transfer occurs leading to
fluorescence at a particular wavelength when the substrates are
excited. Lactamase cleavage of the .beta.-lactam ring is followed
by expulsion of the fluorescein moiety with loss of fluorescence
energy transfer. Excitation of the modified substrate now results
in fluorescence at a different wavelength or results in an increase
in detected fluorescence.
[0067] The assay systems of the present invention further provide
an advantageous and rapid method of isolation and clonal selection
of stably transfected cell lines containing reporter genes and
having the desired properties which the transfection was intended
to confer, e.g. fluorescent signal response after activation of a
transfected receptor with a high signal-to-noise ratio from a high
proportion of isolated cells. Current procedures for clonal
selection of satisfactorily transfected, genetically engineered
cells from the initial population, are done mainly by replica
plating of colonies, testing of one set of colonies, visual
selection of preferred clones, manual isolation of the replicas of
the preferred clones by pipetting, and prolonged cellular
cultivations. This procedure is laborious and time-consuming; it
may require several months to generate a clone useful for assays
suited to drug screening. Moreover, it is difficult to manually
select and maintain more than a few hundred clones. Using the
assays of this present invention, the desired signal from cellular
.beta.-lactamase reporter system can be maintained within living
and viable cells. Replica plating of colonies is unnecessary
because single cells can be assayed and remain viable for further
multiplication. Thus, from the population of initially transfected
cells, one can rapidly select those few individual living cells
with the best fluorescent signal, using automated instruments such
as a fluorescent-activated cell sorter, e.g. the Becton Dickinson
FACS Vantage.TM.. The selected cells are then collected for
cultivation and propagation to produce a clonal cell line with the
desired properties for assays and drug screening.
[0068] As would be immediately apparent to those working in the
field, the combination of a novel substrate in accordance with the
invention and a suitable .beta.-lactamase may be employed in a wide
variety of different assay systems (such as are described in U.S.
Pat. Nos. 4,740,459 and 5,955,604). In particular, the fluorogenic
substrates of the invention enable the detection of
.beta.-lactamase activity in a wide variety of biologically
important environments, such as human blood serum, the cytoplasm of
cells and intracellular compartments; this facilitates the
measurement of periplasmic or secreted .beta.-lactamase.
[0069] Further, the expression of any target protein can be
detected by fusing a gene encoding the target protein to a
.beta.-lactamase gene, which can be localized by immunostaining and
fluorescence or electron microscopy. For example, .beta.-lactamase
fusion proteins may be detected in the lumen of organelles through
the use of the substrates of the invention; only subcellular
compartments containing the fusion protein fluoresce at a
wavelength characteristic of the cleaved substrate, whereas all
others fluoresce at a wavelength characteristic of the intact
molecule.
[0070] Both the intact and cleaved substrate are well retained in
cells without the use of special measures, such as chilling. The
color change (even in individual small mammalian cells) is visible
through a fluorescence microscope using normal color vision or
photographic film; the fluorescence signal may be quantified and
further enhanced by conventional digital image processing
techniques. Moreover, because gene activation is detected not by a
change in a single intensity but rather by a color change or a
change in the ratio between two intensities at different
wavelengths, the assays of the present invention are relatively
immune to many artifacts such as variable leakiness of cells,
quantity of substrate, illumination intensity, absolute sensitivity
of detection and bleaching of the dyes.
[0071] A variety of substrates (e.g., the compounds above and in
Table 1) have been prepared and their emission spectra can be
obtained before and after .beta.-lactamase cleavage. These
substrates allow for .beta.-lactamase detection primarily in vitro,
as they bind strongly to serum and cellular proteins. Due to their
hydrophobic nature, the fluorophores stack; this leads to a loss of
fluorescence in the intact substrate. .beta.-lactamase cleaves the
substrates and relieves the stacking, allowing for
fluorescence.
[0072] The substrates of this invention make it feasible to use
.beta.-lactamase as a reporter gene to monitor the expression from
a set of expression control sequences. In one aspect, this
invention provides methods for monitoring gene expression from a
set of expression control sequences by using .beta.-lactamase as a
reporter gene. A cell is provided that has been transfected with a
recombinant nucleic acid molecule comprising the expression control
sequences operably linked to nucleic acid sequences coding for the
expression of .beta.-lactamase.
[0073] As used herein, the term "nucleic acid molecule" includes
both DNA and RNA molecules. It will be understood that when a
nucleic acid molecule is said to have a DNA sequence, this also
includes RNA molecules having the corresponding RNA sequence in
which "U" replaces "T." The term "recombinant nucleic acid
molecule" refers to a nucleic acid molecule which is not naturally
occurring, and which comprises two nucleotide sequences which are
not naturally joined together. Recombinant nucleic acid molecules
are produced by artificial combination, e.g., genetic engineering
techniques or chemical synthesis.
[0074] Nucleic acids encoding .beta.-lactamases can be obtained by
methods known in the art, for example, by polymerase chain reaction
of cDNA using primers based on the DNA sequence known in the art
and disclosed in U.S. Pat. No. 5,955,604, which is incorporated
herein by reference). PCR methods are described in, for example,
U.S. Pat. No. 4,683,195; Mullis et al. (1987) Cold Spring Harbor
Symp. Quant. Biol. 51: 263; and Erlich, ed., PCR Technology,
(Stockton Press, NY, 1989).
[0075] The construction of expression vectors and the expression of
genes in transfected cells involves the use of molecular cloning
techniques also well known in the art. Sambrook et al., Molecular
Cloning--A Laboratory Manual, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y., (1989) and Current Protocols in Molecular
Biology, F. M. Ausubel et al., eds., (Current Protocols, a joint
venture between Greene Publishing Associates, Inc. and John Wiley
& Sons, Inc., (most recent Supplement)).
[0076] Nucleic acids used to transfect cells with sequences coding
for expression of the polypeptide of interest generally will be in
the form of an expression vector including expression control
sequences operatively linked to a nucleotide sequence coding for
expression of the polypeptide. As used, the term nucleotide
sequence "coding for expression of" a polypeptide refers to a
sequence that, upon transcription and translation of mRNA, produces
the polypeptide. As any person skilled in the are recognizes, this
includes all degenerate nucleic acid sequences encoding the same
amino acid sequence. This can include sequences containing, e.g.,
introns. As used herein, the term "expression control sequences"
refers to nucleic acid sequences that regulate the expression of a
nucleic acid sequence to which it is operatively linked. Expression
control sequences are "operatively linked" to a nucleic acid
sequence when the expression control sequences control and regulate
the transcription and, as appropriate, translation of the nucleic
acid sequence. Thus, expression control sequences can include
appropriate promoters, enhancers, transcription terminators, a
start codon (i.e., ATG) in front of a protein-encoding gene,
splicing signals for introns, maintenance of the correct reading
frame of that gene to permit proper translation of the mRNA, and
stop codons.
[0077] The recombinant nucleic acid can be incorporated into an
expression vector comprising expression control sequences
operatively linked to the recombinant nucleic acid. The expression
vector can be adapted for function in prokaryotes or eukaryotes by
inclusion of appropriate promoters, replication sequences, markers,
etc.
[0078] The recombinant nucleic acid used to transfect the cell
contains expression control sequences operably linked to a
nucleotide sequence encoding a .beta.-lactamase. The
.beta.-lactamase encoded can be any known to the art or described
herein.
[0079] This invention provides novel recombinant nucleic acid
molecules including expression control sequences adapted for
function in a non-mammalian eukaryotic cell operably linked to a
nucleotide sequence coding for the expression of a cytosolic
.beta.-lactamase. As used herein, "cytosolic .beta.-lactamase"
refers to a .beta.-lactamase that lacks amino acid sequences for
secretion from the cell membrane, e.g., the signal sequence.
[0080] It is further preferable that the ribosome binding site and
nucleotide sequence coding for expression of .beta.-lactamase
contain sequences preferred by mammalian cells. Such sequences
improve expression of .beta.-lactamase in mammalian cells.
Preferred sequences for expression in mammalian cells are described
in, for example, Kozak, M., J. Cell Biol. 108.
[0081] When used in mammalian cells, the expression control
sequences are adapted for function in mammalian cells. The method
of this invention is useful to testing expression from any desired
set of expression control sequences. In particular, this invention
is useful for testing expression from inducible expression control
sequences. As used herein, "inducible expression control sequences"
refers to expression control sequences which respond to biochemical
signals either by increasing or decreasing the expression of
sequences to which they are operably linked. For example, in the
case of genes induced by steroid hormones, the expression control
sequences includes hormone response elements. The binding of a
steroid hormone receptor to the response element induces
transcription of the gene operably linked to these expression
control sequences. Expression control sequences for many genes and
for inducible genes, in particular, have been isolated and are well
known in the art. The invention also is useful with constitutively
active expression control sequences.
[0082] The transfected cell is incubated under conditions to be
tested for expression of .beta.-lactamase from the expression
control sequences. The cell or an extract of the cell is contacted
with a .beta.-lactamase substrate of the invention under selected
test conditions and for a period of time to allow catalysis of the
substrate by any .beta.-lactamase expressed. Then the donor moiety
from this sample is excited with appropriate ultraviolet or visible
wavelengths. The degree of fluorescence resonance energy transfer
in the sample is measured.
[0083] If the cell did not express .beta.-lactamase, very little of
the substrate will have been cleaved, the efficiency of FRET in the
cell will be high, and the fluorescence characteristics of the cell
or sample from it will reflect this efficiency. If the cell
expressed a large amount of .beta.-lactamase, most of the substrate
will be cleaved. In this case, the efficiency of FRET is low,
reflecting a large amount or high efficiency of the cleavage enzyme
relative to the rate of synthesis of the tandem fluorescent protein
construct. In one aspect, this method can be used to compare mutant
cells to identify which ones possess greater or less enzymatic
activity. Such cells can be sorted by a fluorescent cell sorter
based on fluorescence.
[0084] Also, as will be apparent to those working in the field of
using reporter gene cell-based assays for screening samples or
pools of samples (such as compounds (combinatorial or synthetic),
natural product extracts, or marine animal extracts) to identify
potential drug candidates which act as agonists, inverse agonists
or antagonists of cellular signaling or activation, the combination
of cells (preferably mammalian) genetically engineered to express
.beta.-lactamase under the control of different regulatory
elements/promoters and the use of the novel .beta.-lactamase
substrate compounds of the present invention will provide distinct
advantages over known reporter genes (including, but not limited
to, chloramphenicol acetyl transferase, firefly luciferase,
bacterial luciferase, vargula luciferase, aequorin,
.alpha.-galactosidase, alkaline phosphatase) and their requisite
substrates.
[0085] By the choice of appropriate regulatory elements and
promoters to control expression of .beta.-lactamase, assays can be
constructed to detect or measure the ability of test substances to
evoke or inhibit functional responses of intracellular hormone
receptors. These include expression control sequences responsive to
inducible by mineralcorticosteroids, including dexamethasone (J.
Steroid Biochem. Molec. Biol. Vol. 49, No. 1 1994, pp. 31),
gluococorticoid, and thyroid hormone receptors (as described in
U.S. Pat. No. 5,071,773). Additional such intracellular receptors
include retinoids, vitamin D3 and vitamin A (Leukemia vol 8, Suppl.
3, 1994 ppS1-S10; Nature Vol. 374, 1995, p. 118-119; Seminars in
Cell Biol., Vol. 5, 1994, p. 95-103). Specificity would be enabled
by use of the appropriate promoter/enhancer element. Additionally,
by choice of other regulatory elements or specific promoters, drugs
which influence expression of specific genes can be identified.
Such drugs could act on specific signaling molecules such as
kinases, transcription factors, or molecules such signal
transducers and activators of transcription (Science Vol. 264,
1994, p. 1415-1421; Mol. Cell Biol., Vol. 16, 1996, p. 369-375).
Specific microbial or viral promoters which are potential drug
targets can also be assayed in such test systems.
[0086] Also by the choice of promoters such as c-fos or c-jun (U.S.
Pat. No. 5,436,128; Proc. Natl. Acad. Sci. Vol. 88, 1991, pp.
5665-5669) or promoter constructs containing regulatory elements
responsive to second messengers (Oncoqene, 6: 745-751 (1991))
(including cyclic AMP-responsive elements, phorbol ester response
element (responsive to protein kinase C activation), serum response
element (responsive to protein kinase C-dependent and independent
pathways) and Nuclear Factor of Activated T-cells response element
(responsive to calcium) to control expression of .beta.-lactamase,
assays can be constructed to detect or measure substances or
mixtures of substances that modulate cell-surface receptors
including, but not limited to, the following classes: receptors of
the cytokine superfamily such as erthyropoietin, growth hormone,
interferons, and interleukins (other than IL-8) and
colony-stimulating factors; G-protein coupled receptors (U.S. Pat.
No. 5,436,128) for hormones, such as calcitonin, epinephrine or
gastrin, pancrine or autocrine mediators, such as stomatostatin or
prostaglandins, and neurotransmitters such as norepinephrine,
dopamine, serotonin or acetylcholine; tyrosine kinase receptors
such as insulin growth factor, nerve growth factor (U.S. Pat. No.
5,436,128). Furthermore, assays can be constructed to identify
substances that modulate the activity of voltage-gated or
ligand-gated ion channels, modulation of which alters the cellular
concentration of second messengers, particularly calcium (U.S. Pat.
No. 5,436,128). Assays can be constructed using cells that
intrinsically express the promoter, receptor or ion channel of
interest or into which the appropriate protein has been genetically
engineered.
[0087] The expression control sequences also can be those
responsive to substances that modulate cell-surface receptors or
that modulate intra-cellular receptors.
[0088] To measure whether a substance or mixture of substances
activates extracellular or intracellular receptors or other
cellular responses, cells containing .beta.-lactamase controlled by
a desired promoter/enhancer element are incubated with test
substance(s), substrate then added, and after a certain period of
time the fluorescence signal is measured at either one or two
excitation-emission pairs appropriate to the chosen compound of the
invention (e.g. compound CCF2 with wavelength pairs of near 405 nm
and near 450 nm and near 405 and near 510 nm). This fluorescent
result is compared to control samples which have had no drug
treatment and, when feasible, control samples with a known
inhibitor and a known activator. The effect of any active drugs is
then determined using the ratio of the fluorescence signal found in
test wells to the signals found in wells with no drug treatment.
Assays are performed in wells in a microtiter plate containing 96
or more wells or in an assay system with no compartments such as a
gel matrix or moist membrane environment. Detection could be done
for example by microtiter plate fluorimeters, e.g. Millipore
Cytofluor, or imaging devices capable of analyzing one or more
wells or one or more assay points in a certain surface area, e.g.
as supplied by Astromed. The ability to retain the substrate in the
cytoplasm of living cells is advantageous as it can allow a
reduction in signal interference from coloured or quenching
substances in the assay medium. Furthermore, the fluorescent signal
from the compounds of this invention, such as CCF2, can be readily
detected in single cells and thus allowing assay miniaturization
and an increased number of tests per surface area. Miniaturized
assays also further increase the throughput of an imaging detection
system as there are more samples within the imaging field.
[0089] The assay systems of the present invention further provide
an advantageous and rapid method of isolation and clonal selection
of stably transfected cell lines containing reporter genes and
having the desired properties which the transfection was intended
to confer, e.g. fluorescent signal response after activation of a
transfected receptor with a high signal-to-noise ratio of at least
10:1 from a high proportion of isolated cells. Current procedures
for clonal selection of satisfactorily transfected, genetically
engineered cells from the population initial transfected with the
vectors of interest, are done mainly by manual means and involve
several rounds of microscopic analyses, selecting the visually
preferred clone, isolation of the clone by manual pipetting stages
and prolonged cellular cultivations. This procedure is laborious
and time-consuming; it may require several months to generate a
clone useful for assays suited to drug screening. Moreover, it is
difficult to manually select and maintain more than a few hundred
clones. Using the assays of this present invention, the desired
signal from cellular .beta.-lactamase reporter system can be
maintained within living and viable cells. Thus, one can rapidly
select, from the population of initially transfected cells, those
few living cells with the best fluorescent signal using automated
instruments such as a fluorescent-activated cell sorter, e.g. the
Becton Dickinson FACS Vantage. The selected cells are then
collected for cultivation and propagation to produce a clonal cell
line with the desired properties for assays and drug screening.
[0090] In addition, the presence (for example, in human serum, pus
or urine) of bacteria resistant to .beta.-lactam antibiotics can be
readily detected using the substrates of the present invention.
Only in the presence of an active .beta.-lactamase is there a
change in the fluorescence spectrum from that of the intact
molecule to one characteristic of the cleavage product. The
substrates of the present invention are superior to prior art
chromogenic substrates Nitrocephin and PADAC, in that the inventive
substrates are stable to human serum. The novel substrates are also
more sensitive than the chromogenic substrate CENTA, because they
experience a much smaller optical background signal from human
serum and a lower detection limit for fluorescence versus
absorbance.
[0091] The invention may be better understood with reference to the
accompanying examples, which are intended for purposes of
illustration only and should not be construed as in any sense
limiting the scope of the invention as defined in the claims
appended hereto.
[0092] The degree of FRET or amount of fluorescence can be
determined by any spectral or fluorescence lifetime characteristic
of the excited construct, for example, by determining the intensity
of the fluorescent signal from the donor, the intensity of
fluorescent signal from the acceptor or quencher, the ratio of the
fluorescence amplitudes near the acceptor's emission maxima to the
fluorescence amplitudes near the donor's emission maximum, or the
excited state lifetime of the donor. For example, cleavage of the
linker increases the intensity of fluorescence from the donor,
decreases the intensity of fluorescence from the acceptor,
decreases the ratio of fluorescence amplitudes from the acceptor to
that from the donor, and increases the excited state lifetime of
the donor.
[0093] Preferably, changes in the degree of fluorescence or FRET
are determined, for example, as a function of the change in the
ratio of the amount of fluorescence from the donor and acceptor
moieties, a process referred to as "ratioing." Changes in the
absolute amount of substrate, excitation intensity, and turbidity
or other background absorbances in the sample at the excitation
wavelength affect the intensities of fluorescence from both the
donor and acceptor approximately in parallel. Therefore the ratio
of the two emission intensities is a more robust and preferred
measure of cleavage than either intensity alone.
[0094] The excitation state lifetime of the donor moiety is,
likewise, independent of the absolute amount of substrate,
excitation intensity, or turbidity or other background absorbances.
Its measurement requires equipment with nanosecond time resolution,
except in the special case of lanthanide complexes in which case
microsecond to millisecond resolution is sufficient.
[0095] Fluorescence in a sample is measured using a fluorimeter. In
general, excitation radiation, from an excitation source having a
first wavelength, passes through excitation optics. The excitation
optics cause the excitation radiation to excite the sample. In
response, fluorescent proteins in the sample emit radiation which
has a wavelength that is different from the excitation wavelength.
Collection optics then collect the emission from the sample. The
device can include a temperature controller to maintain the sample
at a specific temperature while it is being scanned. According to
one embodiment, a multi-axis translation stage moves a microtiter
plate holding a plurality of samples in order to position different
wells to be exposed. The multi-axis translation stage, temperature
controller, auto-focusing feature, and electronics associated with
imaging and data collection can be managed by an appropriately
programmed digital computer. The computer also can transform the
data collected during the assay into another format for
presentation.
[0096] Methods of performing assays on fluorescent materials are
well known in the art and are described in, e.g., Lakowicz, J. R.,
Principles of Fluorescence Spectroscopy, New York:Plenum Press
(1983); Herman, B., Resonance energy transfer microscopy, in:
Fluorescence Microscopy of Living Cells in Culture, Part B, Methods
in Cell Biology, vol. 30, ed. Taylor, D. L. & Wang, Y.-L., San
Diego: Academic Press (1989), pp. 219-243; Turro, N.J., Modern
Molecular Photochemistry, Menlo Park: Benjamin/Cummings Publishing
Col, Inc. (1978), pp. 296-361.
[0097] The Figures and figure legends attached hereto depict assays
based upon the methods and substrates identified above.
[0098] All publications and patent documents cited in this
application are incorporated by reference in their entirety for all
purposes to the same extent as if each individual publication or
patent document were so individually denoted.
[0099] The present invention provides novel substrates for
.beta.-lactamase, .beta.-lactamases and methods for their use.
While specific examples have been provided, the above description
is illustrative and not restrictive. Many variations of the
invention will become apparent to those skilled in the art upon
review of this specification. The scope of the invention should,
therefore, be determined not with reference to the above
description, but instead should be determined with reference to the
appended claims along with their full scope of equivalents.
* * * * *