WO2006100417A1

WO2006100417A1 - Enzyme detection method and reagent

Info

Publication number: WO2006100417A1
Application number: PCT/GB2005/004733
Authority: WO
Inventors: Mark Samuel Jonathan Briggs; Valerie Millar; Nigel Paul Michael
Original assignee: Ge Healthcare Uk Limited
Priority date: 2005-03-22
Filing date: 2005-12-09
Publication date: 2006-09-28
Also published as: GB0505777D0

Abstract

The invention provides a method for determining nitroreductase activity, the method comprising combining a substrate comprising a compound of formula (I): wherein groups Ra and Rb are attached to atoms of the Z ring system; Z represents the a chain of linked atoms necessary to complete an aromatic or heteroaromatic ring system having five or six atoms selected from carbon atoms and optionally no more than two atoms selected from oxygen, nitrogen and sulphur; FLUOR is a fluorescent dye selected from xanthene, coumarin and oxazine dyes; L1 is a bond or is an atom or a chain of 2 to 10 covalently linked atoms selected from the group consisting of carbon, nitrogen, oxygen and sulphur atoms; each Ra is hydrogen or may be selected from electron donating and withdrawing groups, and p is 0 or an integer from 1 to 3; Rb is hydrogen or is the group -L2-W, where L2 is a linker chain containing from 1-20 linked atoms and W is a fluorescent dye moiety or a quenching group; m is 0 or 1; and r is 1 or 2; with a sample containing or suspected to contain nitroreductase under conditions to cause cleavage of FLUOR from the substrate. Nitroreductase activity is determined by detecting a change in an optical property of the sample.

Description

Enzyme Detection Method and Reagent

The present invention relates to an enzymatic method for generating an optical signal from a reporter molecule having a masking moiety attached to the reporter. The invention also relates to masked fluorescent reporter molecules useful as enzyme substrates for the enzyme, nitroreductase.

The use of fluorescence as a detection modality in biological assays is widespread and a diverse variety of procedures are available to generate fluorescence under assay conditions for detection by techniques such as fluorescence microscopy, fluorescence immunoassay and flow cytometry. Among the methods used to generate a fluorescent signal are those which use an enzyme to convert a non-fluorescent or substantially non-fluorescent substrate into a fluorescent product. Such fluorescent enzyme substrates typically have two components that are coupled through a covalent linkage. One component is a fluorescent molecule that is capable of fluorescing by first accepting light energy and then emitting light energy. The other component is a masking group that prevents the fluorescent molecule from accepting or emitting light energy when the two components are covalently bound to one another, such that the molecule is non-fluorescent or substantially non- fluorescent. In the presence of an appropriate enzyme, cleavage of the covalent linkage takes place, thereby allowing the fluorescent molecule to absorb energy and emit fluorescence.

US 5849513 (Jaffe, et al) discloses an assay compound, for assaying the activity inside a metabolically active whole cell. The assay compound includes a leaving group, selected for cleavage by an enzyme to be assayed, and an indicator group. The indicator group is selected from compounds which have a first state when joined to the leaving group, and a second state when the leaving group is cleaved from the indicator group by the enzyme. Preferred indicator compounds are rhodamine 110, rhodol, and fluorescein and analogs of these compounds. US 6207365 (Shiono, et al) describes a method for determining an enzyme by the use of a substrate including a group to be cleaved by an enzyme and a group that forms a strongly fluorescent coumarin derivative through intramolecular lactonization when cleaved by the enzyme. EP 1252520 A (Thomas et al) discloses inter alia a method for detecting nitroreductase enzyme activity by use of a nitro-containing cyanine dye as a substrate for a nitroreductase and measuring an increase in fluorescence signal caused by the enzymatic reduction of the nitro-group in the cyanine dye to a NHOH or NH₂ group by the action of the nitroreductase. Examples of non- fluorescent or substantially non-fluorescent nitro group-containing cyanine dyes for use in the above method are described in US Patent No. 6828116 (Hamilton, A.L. et al) and have formulae (1 ) and (2).

(1 ) (2)

To date however, there are no reports describing the reduction of a masked nitro group-containing dye by the action of a nitroreductase (NTR), in which the masking group is subsequently cleaved from the dye derivative to yield a fluorescent dye molecule. The present invention is concerned with a relatively simple and easily prepared nitro group-containing compound and its use as a masking group for preparing derivatised reporter dyes in which the masking group is covalently attached thereto. When the masking group is covalently attached to the dye, one or more of the optical properties of the dye are modified. For example, a fluorescent dye may be rendered non-fluorescent or substantially non-fluorescent. Following action by a nitroreductase, the nitro group of the masking moiety is reduced to a NHOH or NH₂ group. This action results in cleavage of the dye from the masking group, thereby restoring the optical properties of the dye. The amount of change in the optical property upon such action may be correlated with the amount or activity of the nitroreductase.

In a first aspect of the invention, there is provided a method for determining nitroreductase activity comprising: a) combining a substrate comprising a compound of formula (I):

(D wherein groups R^a and R^b are attached to atoms of the Z ring system;

Z represents the a chain of linked atoms necessary to complete an aromatic or heteroaromatic ring system having five or six atoms selected from carbon atoms and optionally no more than two atoms selected from oxygen, nitrogen and sulphur; FLUOR is a fluorescent dye selected from xanthene, coumarin and oxazine dyes; Li is a bond or is an atom or a chain of 2 to 10 covalently linked atoms selected from the group consisting of carbon, nitrogen, oxygen and sulphur atoms; each R^a is hydrogen or may be selected from electron donating and withdrawing groups, and p is 0 or an integer from 1 to 3; R^b is hydrogen or is the group -L₂-W, where L₂ is a linker chain containing from 1- 20 linked atoms and W is a fluorescent dye moiety or a quenching group; m is 0 or 1 ; and r is 1 or 2; with at least a portion of a sample containing or suspected to contain nitroreductase under conditions to cause cleavage of FLUOR from said substrate; and b) detecting a change in an optical property of said sample following the combining step a); wherein said change is a measure of the amount of nitroreductase activity. Suitably, in the first aspect of the invention, the sample in which nitroreductase enzyme activity is to be determined comprises a tissue sample or a cell extract. Alternatively, the sample may comprise at least one cell, in which case suitably, the enzyme substrate is, or is rendered, cell permeable. In principle, any type of cell can be used, including all normal and transformed cells derived from any recognised source with respect to species (e.g. human, rodent, simian), tissue source (e.g. brain, liver, lung, heart, kidney skin, muscle) and cell type (e.g. epithelial, endothelial); plant, fungal and bacterial cell types. Where appropriate, a cell extract can be prepared from a cell, using standard methods known to those skilled in the art (Molecular Cloning, A Laboratory Manual 2^nd Edition, Cold Spring Harbour Laboratory Press 1989), prior to measuring fluorescence. Typical conditions for nitroreductase activity comprise incubation of the sample containing or suspected to contain nitroreductase in a suitable medium and the substrate at approximately 37⁰C in the presence of NADH and FMN.

In one embodiment according to the first aspect, the method allows screening for a test agent whose effect upon the activity of a nitroreductase is to be determined. The method comprises the steps of: (a) performing the method according to the first aspect in the presence and in the absence of the test agent; and (b) determining the activity of the enzyme in the presence and in the absence of the test agent. A difference between the activity of the nitroreductase enzyme in the presence and in the absence of the said agent is indicative of the effect of the agent on the activity of the enzyme.

In the alternative, the method of screening for a test agent may be performed according to the first aspect in the presence of said agent, and comparing the value of the activity of the enzyme with a control value for the enzyme activity in the absence of the test agent. The control value may be stored electronically in a database or other electronic format.

In a second aspect of the invention, there is provided a method for detecting nitroreductase gene expression, said method comprising the steps of: a) providing a host cell wherein said host cell has been transfected with a nucleic acid molecule comprising expression control sequences operably linked to a sequence encoding a nitroreductase; b) contacting said host cell with a substrate under conditions to promote nitroreductase activity and wherein said substrate comprises a compound of formula (I):

(D wherein groups R^a and R^b are attached to atoms of the Z ring system; Z represents the a chain of linked atoms necessary to complete an aromatic or heteroaromatic ring system having five or six atoms selected from carbon atoms and optionally no more than two atoms selected from oxygen, nitrogen and sulphur; FLUOR is a fluorescent dye selected from xanthene, coumarin and oxazine dyes; Li is a bond or is an atom or a chain of 2 to 10 covalently linked atoms selected from the group consisting of carbon, nitrogen, oxygen and sulphur atoms; each R^a is hydrogen or may be selected from electron donating and withdrawing groups, and p is 0 or an integer from 1 to 3; R^b is hydrogen or is the group -L₂-W, where L₂ is a linker chain containing from 1- 20 linked atoms and W is a fluorescent dye moiety or a quenching group; m is 0 or 1 ; and r is 1 or 2; and c) detecting a change in an optical property upon cleavage of said FLUOR from said substrate; wherein said change is a measure of the amount of nitroreductase gene expression.

The optical property that is detected is suitably the intensity of emitted fluorescence, as a result of the action of the nitroreductase upon, and cleavage of the fluorescent dye moiety from the substrate. For example, the fluorescence emission intensity of the substrate may be determined in the absence of nitroreductase upon excitation of the FLUOR moiety and/or W at its typical excitation wavelength. Following combination of the substrate with nitroreductase enzyme, the fluorescence emission intensity is again measured at the emission wavelength of FLUOR and/or W and the change in measured fluorescence is determined. Suitably, the change in fluorescence may be either an increase or a decrease in fluorescence intensity. Measurements of the amount of nitroreductase activity may be either quantitative (and thereby correlated to the amount of nitroreductase present), or the measure may be qualitative and be used to determine the presence or absence of nitroreductase. Excitation of the nitroreductase substrate and measurement of fluorescence emission may also be performed over a range of wavelengths, so as to maximise emission signal and to distinguish between excitation and emission signals. Alternatively, the measured change in an optical property may be a change in fluorescence lifetime of the dye, before and after the action of the nitroreductase upon the composition. The change in fluorescence lifetime may also be used to distinguish the product of the enzyme reaction from the dye molecule used as the substrate. As a further alternative, the change in an optical property may be a change in the absorption maximum of the dye molecule, relative to the absorption maximum of the product. In preferred embodiments, the change in an optical property is an increase in the fluorescence intensity of the dye molecule, whereby the increase is a measure of the amount of nitroreductase activity.

In one embodiment, L₁ is a single covalent bond linking the mono-nitro group-containing aralkyl or aralkenyl masking group and the fluorescent dye. Alternatively, Li is an atom or a chain of 2 to 10 branched or unbranched covalently linked atoms linking the masking group and dye and is selected from the group consisting of carbon, nitrogen, oxygen and sulphur atoms. For example, L₁ may comprise one or more atoms or groups selected from -CHR¹- , -NR'-, -O- -S-, -C(O)- and -C(S)-, where R' is hydrogen or C₁-C₄ alkyl. Examples of suitable linking groups L₁ include: -0-0(0)-, -0-C(O)-O-, -0-C(O)-NH-, and -0-C(O)-NH-CH₂-.

Suitably, Z in the compound of formula (I) is selected from phenyl, naphthyl, imidazolyl, oxazolyl and thiazolyl moieties. Preferably Z is a phenyl or an imidazolyl ring system. Preferably, the masking group attached to FLUOR are selected from:

wherein R^a, R^b, R^c, Li and p are hereinbefore defined, R^c is hydrogen or C₁-C₄ alkyl and m is O.

Preferably, the substrate is a compound of formula:

wherein Li and FLUOR are hereinbefore defined.

In one embodiment, FLUOR is selected from xanthene dyes including fluorescein derivatives, rhodamine derivatives and rhodol derivatives. In another embodiment, FLUOR is a coumarin dye. In a still further embodiment, FLUOR is an oxazine dye.

Suitably, one or more of groups R^a are selected from electron donating and withdrawing groups, for example, cyano, halogen, hydroxyl, Ci-C₄ alkyl, -NO₂, -NHCO₂R^d, -CO₂H, -CO₂R^d, -SH, Ci-C₄ alkylamino, and C₁-C₄ alkoxyl; where R^d is C₁-C₄ alkyl. Preferred groups R^a are either hydrogen or electron donating groups selected for their ability to enhance the rate of cleavage of the masking group from FLUOR, for example, C₁-C₄ alkyl, Ci-C₄ alkoxyl and C1-C₄ alkylamino, preferably methyl, methoxyl, or methylamino. Halogen and halo groups may be selected from fluoro, chloro, bromo and iodo.

In one embodiment, in the compound of formula (I), R^b is hydrogen. In another embodiment, R^b is the group -Lr-W, where L2 and W are hereinbefore defined. Preferably, linker chain L₂ contains from 1 - 10 linked atoms. The linker chain may include part of the constituents extending from the fluorochrome. In other words, the linker is attached to the dye chromophore but is not a part of it. When R^b is the group -L₂-W, preferably, FLUOR and W are linked such that under suitable conditions the optical properties of said compound are modified, for example energy transfer (ET) may take place one with the other. One mode of energy transfer is fluorescence resonance energy transfer (FRET), which is a distance-related process in which the electronic excited states of two dye molecules interact without emission of a photon. See, Forster, T., "Intermolecular Energy Transfer and Fluorescence", Ann. Physik., Vol.2, p.55, (1948). One result of this interaction is that excitation of a donor molecule enhances the fluorescence emission of an acceptor molecule. The fluorescence quantum yield of the donor is correspondingly diminished. For FRET to occur, suitably, the donor and acceptor dye molecules must be in close proximity one with the other, since energy transfer efficiency decreases inversely as the 6^th power of the distance between the donor and acceptor molecules. Preferably, the distance between donor and acceptor is in the range between 10-100 Angstroms. Suitably, in the present invention, either FLUOR or W may be the donor or acceptor in the FRET relationship such that when FLUOR is the donor molecule, W is the acceptor and vice versa. By donor, it is meant that the dye moiety is capable of absorbing energy from light and emits light at wavelength frequencies which are at least partly within the absorption spectrum of the acceptor. By acceptor, it is meant that the dye moiety is capable of absorbing energy at a wavelength emitted by a donor dye moiety. Suitably, there is overlap between at least a portion of the emission spectrum of the donor dye molecule with the absorption spectrum of the acceptor dye molecule. Suitably, the wavelength of the emission maximum of the acceptor dye is longer that the wavelength of the emission maximum of the donor dye. In one format, one of the donor or acceptor molecules can be a quenching group which is in close proximity to a second fluorescent acceptor or donor fluorophore. Upon excitation of the non-fluorescent dye, energy is dissipated as heat rather than fluorescence energy and resonance energy transfer or fluorescence emission cannot take place. In either format, a change in fluorescence signal will be observed when the FRET pair is released from close proximity, i.e. by cleavage of the masking group linking the two chromophores.

When FLUOR is the donor dye and W is the acceptor dye, in a first fluorescence state, excitation of the cassette at the normal excitation wavelength of FLUOR will result in minimal emission of fluorescence signal at the emission wavelength of FLUOR. Action of a nitroreductase upon the FRET cassette results in cleavage of the masking group from FLUOR. Excitation of FLUOR will result in emission of fluorescence at the emission wavelength of FLUOR. Thus, an increase in fluorescence signal will be detected.

When W is the donor dye and FLUOR is the acceptor dye, then in a first fluorescence state, excitation of the FRET cassette at the normal excitation wavelength of W will result in emission of a fluorescence signal at the normal emission wavelength of FLUOR. Upon action of a nitroreductase, the FRET cassette will be cleaved, thereby separating FLUOR and W. Excitation of W at its excitation wavelength will result in a reduced fluorescence emission signal from FLUOR measured at its emission wavelength.

When FLUOR is the donor dye and W is a quenching group then, in a first fluorescence state, excitation of the cassette at the excitation wavelength of FLUOR will result in no, or substantially no fluorescence emission from FLUOR. Upon action by a nitroreductase, cleavage of the masking group takes place and excitation of FLUOR at its excitation wavelength will result in an increase in fluorescence signal at the emission wavelength of FLUOR. Suitably, FLUOR may be selected from xanthene dyes (including their tautomeric forms), for example fluoresceins, rhodamines, rhodols and their derivatives; coumarin dyes; benzocoumarin dyes; and oxazine dyes. Suitable fluorescein dye derivatives will be well known to the skilled person and include but are not limited to fluorescein, 5-carboxyfluorescein, 6-carboxyfluorescein, 6- carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein, 5-(and-6)-carboxyfluorescein, 6-carboxy-2',4,4',5',7,7'- hexachlorofluorescein,

tetrachlorofluorescein, 2',7'-difluorofluorescein and eosin. Suitable rhodamine dyes include but are not limited to: 5-carboxyrhodamine (Rhodamine 110-5), 6- carboxyrhodamine (Rhodamine 110-6), 5-carboxyrhodamine-6G (R6G-5 or REG-5), 6-carboxyrhodamine-6G (R6G-6 or REG-6), N,N,N',N'-tetramethyl-5- carboxyrhodamine, N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA or TMR), 5-carboxy-X-rhodamine and 6-carboxy-X-rhodamine (ROX). Xanthene dyes suitable for conjugation to the masking group are available commercially (see for example, "Handbook of Fluorescent Probes and Research Chemicals", published by Molecular Probes Inc.

Suitable coumarin dyes include, but are not limited to, 7-hydroxy- coumarin, 7-hydroxycoumarin-4-acetic acid, 7-hydroxy-4-methylcoumarin, 7- amino-4-methylcoumarin and 7-amino-4-(trifluoromethyl)coumarin. Additional coumarin derivatives will be well known to the skilled person. Suitable oxazine dye derivatives include resorufin, Nile Blue and cresyl violet.

When W is selected to be a fluorescent dye, it may be selected from any suitable dye, with the proviso that W- is either a donor or an acceptor in a FRET relationship with FLUOR. Example of such dyes include coumarin dyes, benzocoumarin dyes, phenoxazine dyes, xanthene dyes, cyanine dyes and derivatives of the bis-pyrromethine boron difluoride dyes, such as 3, 3', 5, 5'- tetramethyl-2,2'-pyrromethene-1 ,1 '-boron difluoride, sold under the trademark BODIPY by Molecular Probes Inc. When W is a quenching group, it may be selected from groups such as 2,4-dinitrophenyl (DNP) and 4-(4- dimethylaminophenyl)-azobenzoic acid (DABCYL), Malachite green, QSY 7, QSY 21 , QSY 35, methyl red, methyl orange and Black Hole Quencher™. For the determination of nitroreductase activity in cellular samples, it is advantageous that the fluorescence of the substrate be measured at wavelengths above about 500nm, thereby avoiding background fluorescence caused by components of the cells.

Methods for detecting and measuring nitroreductase gene expression in cell based assays are becoming increasingly attractive compared with in vitro biochemical assays and may be use in high throughput screening (HTS) applications. This is because cell based assays require minimal manipulation and the readouts can be examined in a biological context that more faithfully mimics the normal physiological situation. Such in vivo assays require an ability to measure a cellular process and a means to measure its output. For example, a change in the pattern of transcription of a number of genes can be induced by cellular signals triggered, for example, by the interaction of an agonist with its cell surface receptor or by internal cellular events such as DNA damage. The induced changes in transcription can be identified by fusing a reporter gene to a promoter region which is known to be responsive to the specific activation signal. In fluorescence-based enzyme-substrate systems, an increase in fluorescence gives a measure of the activation of the expression of the reporter gene.

For use as a reporter gene, the nitroreductase gene may be isolated by well known methods, for example by amplification from a cDNA library by use of the polymerase chain reaction (PCR) (Molecular Cloning, A Laboratory Manual 2^nd Edition, Cold Spring Harbour Laboratory Press 1989 pp 14.5-

14.20). Once isolated, the nitroreductase gene may be inserted into a vector suitable for use with mammalian promoters (Molecular Cloning, A Laboratory Manual 2^nd Edition, Cold Spring Harbour Laboratory Press 1989 pp 16.56- 16.57) in conjunction with and under the control of the gene regulatory sequence under study. The vector containing the nitroreductase reporter and associated regulatory sequences may then be introduced into the host cell by transfection using well known techniques, for example by use of DEAE-Dextran or Calcium Phosphate (Molecular Cloning, A Laboratory Manual 2^nd Edition, Cold Spring Harbour Laboratory Press 1989 pp 16.30-16.46). Other suitable techniques will be well known to those skilled in the art.

In one embodiment according to the second aspect, the assay method is conducted in the presence of a test agent whose effect on gene expression is to be determined. Typically, to assay the activity of a test agent to modulate a cellular response, cells transfected with the nitroreductase reporter gene are incubated with the test agent, followed by addition of a nitroreductase substrate of formula (I). Suitably, the nitroreductase substrate is, or is rendered, permeable to cells under investigation.

The method comprises the steps of: a) performing the method according to the second aspect in the presence and in the absence of the test agent; and b) determining the amount of nitroreductase gene expression in the presence and in the absence of said agent. Measurement of a difference in nitroreductase gene expression in the presence and in the absence of the agent is indicative of the effect of the test agent on nitroreductase gene expression. Alternatively, the screening can be done by performing the method in the presence of a test agent and comparing the value of the activity of the enzyme with a control value for the enzyme activity in the absence of the test agent. The control value may be conveniently stored electronically in a database or other electronic format. After an appropriate period required for cleavage of the nitroreductase substrate and liberation of the FLUOR moiety, the fluorescence from the cells is measured at an emission wavelength appropriate for the chosen dye.

Suitable means for expressing a nitroreductase enzyme include an expression plasmid or other expression construct. Methods for preparing such expression constructs are well known to those skilled in the art.

A test agent may be, for example, any organic or inorganic compound such as a synthetic molecule or a natural product (e.g. peptide, oligonucleotide), or a may be an energy form (e.g. light or heat or other forms of electromagnetic radiation). Suitably, the difference between the activity of the enzyme in the absence and in the presence of the agent is normalised, stored electronically and compared with a reference value. Thus, for example, the difference in activity may be stored as a percentage inhibition (or percentage stimulation) on an electronic database and this value compared with the corresponding value for a standard inhibitor of the enzyme in question. In this way, only test agents meeting a certain pre-determined threshold (e.g. as being as effective or more effective than the reference compound) may be selected as being of interest for further testing.

Typically, cultured cells are incubated with the NTR substrate at a concentration of 0.1 to 100μM in a suitable cell culture medium under conditions suitable for cell growth and for a time which may range from 0.5 to 24 hours. Cells are cultured according to standard cell culture techniques, e.g. cells are cultured in a suitable vessel in a sterile environment at 37⁰C in an incubator containing a humidified 95% air/5% CO₂ atmosphere. There are established protocols available for the culture of diverse cell types. (See for example, Freshney, R. I., Culture of Animal Cells: A Manual of Basic Technique, 2^nd Edition, Alan R.Liss Inc. 1987). When the substrate is required to be introduced into cells grown in cell or tissue culture, the substrate is simply added to the culture medium. Cells may also be contacted with the substrate in the presence of a test agent whose effect on the enzyme activity is to be determined. In this embodiment, the detection step provides a measurement of the effect of the test agent on the activity of the enzyme under investigation.

Measurements of fluorescence intensity may be made using instruments incorporating photo-multiplier tubes as detectors. Changes in fluorescence intensity may also be measured by means of a charge coupled device (CCD) imager (such as a scanning imager or an area imager) to image all of the wells of a microtitre plate. The LEADseeker™ system features a CCD camera allowing fluorescence imaging of high density microtitre plates in a single pass. Imaging is quantitative and fast, and instrumentation suitable for imaging applications can now simultaneously image the whole of a multiwell plate. Where an assay is to be formatted for the determination of the activity of a test agent on enzyme activity, the assay may be performed under continuous measurement of the fluorescence of the substrate. In this format, the intensity of the fluorescent labelled substrate changes continuously. The labelled substrate does not need separation from the product of the enzymatic reaction and thus, a time-course of the reaction may be obtained, allowing kinetic studies to be performed in real time. The measured fluorescence may be compared with fluorescence from control cells not exposed to the test agent and the effects, if any, of the test agent on gene expression modulated through the regulatory sequence is determined by the detection of the characteristic fluorescence in the test cells. Where appropriate, a cell extract can be prepared using conventional methods.

In a third aspect of the present invention, there is provided a test kit for measuring the nitroreductase gene expression. Suitably, the test kit comprises a reporter system comprising means for expressing a nitroreductase enzyme and a compound of formula (I). Preferably, means for expressing a nitroreductase enzyme comprises a nucleic acid comprising expression control sequences operably linked to a sequence encoding a nitroreductase and being optionally to a target gene of interest.

In a fourth aspect, the present invention also provides masked reporter dyes of formula (I), which may be employed as nitroreductase substrates in the methods according to the present invention. In one embodiment, FLUOR is a xanthene dye, (including fluoresceins, rhodamines and rhodols and their tautomeric forms), whereupon the nitroreductase substrate is suitably of the formula:

wherein X is OR¹² or NR¹²R¹³ and Y is O or N⁺R¹²R¹³; R¹, R², R³, R⁴, R⁵, R⁶ and R⁷ are selected from hydrogen, halogen, sulphonic acid, sulphonate, cyano, C₁-C₄ alkyl, Ci-C₄ perfluoro and Ci-C₄ alkoxyl; at least one of groups R⁸, R⁹, R¹⁰, R¹¹, R¹² and R¹³ is the group:

wherein groups R^a, R^b, Z, p and m are hereinbefore defined; Li is a bond or is chain of covalently linked atoms selected from:

-O-C(O)-,

-0-C(O)-O-,

-0-C(O)-NH-, and

-0-C(O)-NH-CH₂- ; remaining groups R⁸, R⁹ and R¹⁰ are independently selected from hydrogen halogen, carboxyl, sulphonic acid, sulphonate, cyano, Ci-C₄ alkyl, Ci-C₄ perfluoro and Ci-C₄ alkoxyl; remaining groups R¹¹, R¹², and R¹³ are independently selected from hydrogen and Ci-C₄ alkyl or when Y is N⁺R¹²R¹³, R¹² taken in combination with R¹ and R¹³ taken in combination with R², together form a fused tricyclic ring system each ring containing 6 atoms; and remaining group R¹¹ is hydrogen or Ci-C₄ alkyl, preferably methyl or ethyl.

In another embodiment, FLUOR is a coumarin dye derivative and the substrate is suitably of the formula:

wherein X is OR¹² or NR¹²R¹³; at least one of groups R¹² and R¹³ is the group:

wherein groups R^a, R^b, Z, p and m are hereinbefore defined; L₁ is a chain of covalently linked atoms selected from:

-O-C(O)-,

-0-C(O)-Q-,

-0-C(O)-NH-, and

-0-C(O)-NH-CH₂- ; remaining group R¹²or R¹³ is selected from hydrogen and Ci-C₄ alkyl; and R¹⁴ is selected from Ci-C₄ alkyl, CF₃, CO₂H and CH₂CO₂H.

In a still further embodiment, FLUOR is an oxazine dye derivative, and the substrate is suitably of the formula:

wherein X is OR¹² or NR¹²R¹³ and Y is O or N⁺R¹²R¹³; wherein at least one group R¹² or R¹³ is the group:

O,N_V

Rb^-

-^{' '} wherein groups R^a, R , Li, Z, p and m are hereinbefore defined; and remaining groups R¹² and R¹³ are selected from hydrogen and Ci-C₄ alkyl. Examples of masked dye molecules that may be used in methods of the present invention are shown in Table 1.

Table 1 Examples of Masked Fluorescent Dyes

(II) (III)

(IV)

(V) Table 1 (continued)

(Vl)

In preferred embodiments, the nitroreductase substrate employed in the methods according to the present invention is, or is rendered, permeable to cells. In these embodiments, at least one of groups Rⁿ comprises a cell membrane permeabilising group, where n is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 and 14. Membrane permeant compounds can be generated by masking hydrophilic groups of the dye moiety to provide more hydrophobic compounds. The masking groups can be designed to be cleaved from the substrate within the cell to generate the derived substrate intracellularly. Since the substrate is more hydrophilic than the membrane permeant derivative, it is trapped within the cell. Suitable cell membrane permeabilising groups may be selected from acetoxymethyl ester which is readily cleaved by endogenous mammalian intracellular esterases (Jansen, A. B. A. and Russell, T. J., J. Chem. Soc. 2127- 2132 (1965) and Daehne, W. et al. J. Med-. Chem. 13, 697-612 (1970)) and pivaloyl ester (Madhu et al., J. Ocul. Pharmacol. Then 1998, 14, 5, pp 389- 399), although other suitable groups will be recognised by those skilled in the art. The invention also provides a nitro group-containing compound for use as a reagent for preparing a nitroreductase enzyme substrate. Thus, in a fifth aspect there is provided use of a reagent for preparing a masked reporter dye according to formula (I),

(I) wherein groups R^a and R^b are attached to atoms of the Z ring system; Z represents the a chain of linked atoms necessary to complete an aromatic or heteroaromatic ring system having five or six atoms selected from carbon atoms and optionally no more than two atoms selected from oxygen, nitrogen and sulphur; FLUOR is a fluorescent dye; Li is a bond or is an atom or a chain of 2 to 10 covalently linked atoms selected from the group consisting of carbon, nitrogen, oxygen and sulphur atoms; each R^a is hydrogen or may be selected from electron donating and withdrawing groups, and p is 0 or an integer from 1 to 3; R^b is hydrogen or is the group -L₂-W, where L₂ is a linker chain containing from 1 - 20 linked atoms and W is a fluorescent dye moiety or a quenching group; m is 0 or 1 and r is 1 or 2. The reagent is a compound having the formula (II):

(H) wherein groups R^a, R^b, Z, L-i, p and m are hereinbefore defined and Q is a reactive group. In principle, any fluorescent dye may be derivatised by reaction of the dye with a compound according to formula (II). Suitable fluorescent dyes may be selected from xanthene dyes, coumarin dyes, oxazine dyes and cyanine dyes. Particularly preferred are xanthene (including fluorescein and derivatives, rhodamine and derivatives), coumarin and oxazine dyes.

Suitably, one or more of groups R^a are selected from electron donating and withdrawing groups, for example, cyano, halogen, hydroxyl, C₁-C₄ alkyl, -NO₂, -NHCO₂R^d, -CO₂H₁ -CO₂R^d, -SH, C₁-C₄ alkylamino, and C₁-C₄ alkoxyl; where R^d is C₁-C₄ alkyl. Preferred groups R^a are either hydrogen or electron donating groups selected for their ability to enhance the rate of cleavage of the masking group from FLUOR, for example, C₁-C₄ alkyl, C₁-C₄ alkoxyl and C₁-C₄ alkylamino, preferably methyl, methoxyl, or methylamino.

Suitably, Q is a group chosen so as to be reactive with a complementary functional group of the fluorescent dye. Typically, Q is a leaving group selected from halogen, (for example chloro, bromo or iodo), Ci-C₄ alkoxy, for example methoxy and ethoxy, O-mesylate and O-triflate.

The NTR substrates are conveniently prepared by reacting a suitable nitro aryl compound with a selected dye in a suitable solvent medium and in the presence of a base. For example, 4-nitro benzyl bromide may be reacted with fluorescein in DMF in the presence of potassium carbonate, with displacement of bromide, to give the nitro aryl-dye conjugate. For the synthesis of the cassettes, a stepwise synthesis may be required, for example a suitable nitro aryl starting material such as 6-nitro phthalide may first be reacted with a dye (or a spacer moiety and then a dye), and subsequently conjugated to a second dye (where the first and second dyes may be the same or different).

The invention is further illustrated by reference to the following examples and figures, in which:

Figure 1 shows the fluorescence intensity change upon the reaction of fluorescein 4-nitrobenzyl ether with nitroreductase with respect to time, and using suitable controls, as follows: D nitroreductase not present (control experiment), ■ nitroreductase (and necessary co-factor e.g. NADH) present, V nitroreductase substituted with BSA (control experiment), 0 nitroreductase present, but co-factor NADH not present (control experiment); Figure 2 shows the fluorescence intensity change upon the reaction of fluorescein 4-nitrobenzyl ester with nitroreductase with respect to time, using suitable controls: D nitroreductase not present (control experiment), ■ nitroreductase (and necessary co-factor e.g. NADH) present, V nitroreductase substituted with BSA (control experiment), Φ nitroreductase present, but co- factor NADH not present (control experiment); Figure 3 shows the fluorescence intensity change upon the reaction of fluorescein bis-4-nitrobenzyl ether with nitroreductase with respect to time, using suitable controls: D nitroreductase not present (control experiment), ■ nitroreductase (and necessary co-factor e.g. NADH) present, V nitroreductase substituted with BSA (control experiment), 0 nitroreductase present, but co-factor NADH not present (control experiment); Figure 4 shows the fluorescence intensity change upon the reaction of resorufin 4-nitrobenzyl carbonate with nitroreductase with respect to time, using suitable control: ▲ nitroreductase (and necessary co-factor e.g. NADH) present

♦ nitroreductase not present (control experiment);

Figure 5 shows the fluorescence intensity change upon the reaction of the fluorescein-coumarin cassette with nitroreductase with respect to time;

Figure 6 shows the fluorescence intensity change upon the reaction of the Cy3- Cy5 cassette with nitroreductase with respect to time: ■ Cy3™ (excitation 540nm, emission 562nm), ACy5 (excitation 540nm, emission 670nm); Figure 7 shows the fluorescence intensity change upon the exposure of fluorescein 4-nitrobenzyl ester to cells expressing nitroreductase (as modulated by the addition of TNFα) with respect to time, together with a suitable control. CytoCyδ is also depicted for direct comparison: Δ fluorescein 4-nitro benzyl ester control (- TNFα), A fluorescein 4-nitro benzyl ester stimulated (+ TNFα), o CytoCyδ control (- TNFα), • CytoCyδ stimulated (+ TNFα); Figure 8 shows the fold induction of fluorescein 4-nitrobenzyl ester versus

CytoCyδ according to Example 9: T Fluorescein 4-nitrobenzyl ester, •

CytoCyδ;

Figure 9 shows the signal:noise ratio of fluorescein 4-nitrobenzyl ester versus

CytoCyδ according to Example 9: ■ CytoCyδ, D Fluorescein 4-nitrobenzyl ester.

Black hole quencher is a trademark of Biosearch Technologies, Inc. Cy and LEADseeker are trademarks of GE Healthcare Limited.

Examples

1. Preparation of fluorescein bis-4-nitrobenzyl ether

Fluorescein (664mg, 2.0mmol), 4-nitrobenzy bromide (648mg, 3.0mmol) and silver (I) oxide (718mg, 3.1 mmol) were suspended in dry toluene (80ml) under a dry nitrogen atmosphere. The mixture was heated at reflux for approximately 7 days during which time the volume decreased to approximately 20ml. The mixture was then cooled to room temperature, diluted with THF (100ml) and filtered through celite. The filter cake was washed with THF and the combined filtrates were concentrated in vacuo and the residue triturated with hot dichloromethane. The dark red-brown supernatant was separated, concentrated in vacuo and then redissolved in a dichloromethane:hexane mixture. Upon cooling and storage at -2O⁰C an orange-beige precipitate formed that was collected and re-precipitated from hot toluene:acetone, washed with acetone and dried to afford the title compound.

δH (270MHz; CDCI3) 5.2 (4H₁ s), 6.7 (4H, m), 6.8 (2H, m), 7.2 (1H, m), 7.6 (6H, m), 8.0 (1 H₁ m), 8.3 (4H₁ m).

Ettan LCMS: C₃4H₂2N₂O9 requires 602. Found MH⁺ = 603.

2. Preparation of fluorescein 4-nitrobenzyl ether and fluorescein 4- nitro benzyl ester

Fluorescein 4-nitrobenzyl ether Fluorescein 4-nitrobenzyl ester

Fluorescein (950mg, 2.85mmol) and potassium carbonate (394mg, 2.85mmol) were suspended in dry DMF (6ml) to give a dark red-brown mixture. After 15min. a solution of 4-nitrobenzyl bromide (713mg, 3.3mmol) in dry DMF (3ml) was added drop wise. The mixture was stirred for a further 2.5hr in the dark and then poured into rapidly stirred 5M hydrochloric acid (50ml). The precipitate that formed was collected by filtration and washed with water (200ml). The collected solids were then redissolved in dichloromethane and washed with 50% saturated sodium bicarbonate (aq) (100ml), water (100ml), 5M hydrochloric acid (50ml) and brine (50ml). The organic phase was dried over MgSO₄ and concentrated in vacuo. This material was then combined with that from a second preparation that was performed on a similar scale but at a lower temperature (ice bath) and worked up by pouring into water (200ml) and collecting the orange precipitate that formed. Flash silica column chromatography (dichloromethane to 10% methanol in dichloromethane gradient) afforded the title compounds.

Fluorescein 4-nitrobenzyl ether: δH (270MHz; DMSO) 5.4 (2H, s), 6.6 (2H, m), 6.7 (2H, m), 6.8 (1 H, m), 7.0 (1 H₁ m), 7.3 (1 H, m), 7.7 (4H, m), 8.0 (1 H, m), 8.3 (2H₁ m). Ettan LCMS: C₂₇Hi₇NO₇ requires 467. Found MH⁺ = 468.

Fluorescein 4-nitrobenzyl ester: δH (270MHz; DMSO) 5.1 (2H, s), 6.3 (2H, m), 6.5 (2H, m), 6.7 (2H, m), 7.3 (2H, m), 7.5 (1 H, m), 7.8 (2H, m), 8.0 (2H, m), 8.2 (1 H₁ m). Ettan LCMS: C₂₇Hi₇NO₇ requires 467. Found MH⁺ = 468.

3. Evaluation of fluorescein derivatives as reagents for detecting nitroreductase activity

The following standard protocol was used in order to test the fluorescein derivatives described above. The assays were performed in microtitre plates which were subsequently counted on a Tecan Ultra plate reader using standard fluorescein filter sets. Total assay volume per well (200μl) was as shown in Table 2 below:

Table 2

The above reagents were pipetted into a 96-well, black microtitre plate. The assay mix was then incubated at room temperature and the plates read on the fluorescent plate reader at timed intervals. Three control sets of experiments were used: firstly the absence of NTR, secondly the substitution of NTR with BSA and thirdly in the absence of NADH. In all cases, a significant increase in fluorescence signal intensity at fluorescein emission wavelengths was observed, relative to controls (see Figures 1 , 2 and 3).

4. Preparation of resorufin 4-nitrobenzyl carbonate

Resorufin sodium salt (235mg, 1.Ommol) was suspended in dry DMF

(5ml) under a dry nitrogen atmosphere. 4-Nitrobenzylchloroformate (259mg, 1.2mmol), as a solution in dry DMF, was then added drop wise over 5 min. The reaction mixture was stirred at room temperature for a further 8 hrs after which it was partitioned between water and dichloromethane. The aqueous phase was extracted with dichloromethane (3x50ml) and the combined organic phase was washed repeatedly with water until the washings were virtually colourless. The organic phase was dried over MgSO₄ and filtered. The filtrate was allowed to stand at room temperature overnight and then concentrated in vacuo to give an orange brown residue. The residue was then triturated with diethyl ether (25ml), centhfuged and the supernatant removed. This process was repeated to give a solid that was stored at room temperature for approximately 64 hrs and then dried in vacuo at 4O⁰C to give an orange powder (237mg).

δH (270MHz; DMSO) 5.5 (2H, s), 6.3 (1 H, m), 6.8 (1 H, m), 7.4 (1H, m), 7.6 (2H₁ m), 7.8 (2H, m), 7.9 (1 H, m), 8.3 (2H, m). 5. Evaluation of resorufin 4-nitrobenzyl carbonate as a reaαent for detecting nitroreductase activity

Enzyme studies were performed using the following standard procedure. Into a cuvette was added: i) Phosphate buffered saline (PBS) pH7.4 (1790μl) ii) NADH (200μl of a 5mg in 640μl PBS solution) iii) Dye (4μl of a 1 mM DMSO solution)

Fluorescence intensity was measured (T = 0) on a Perkin-Elmer LS55B fluorometer using the following instrument parameters: excitation 480nm (slit width 5nm), emission 584nm (slit width 5nm). NTR enzyme (5μl of 5.5ng/ml PBS solution) was added (the enzyme was previously saturated with FMN co- factor to ensure maximum activity). Fluorescence intensity was then measured over time. A control experiment was run sequentially, in which the NTR enzyme was substituted with buffer. During this period there was a noticeable visual colour change of the assay mixture containing NTR from a very pale yellow-brown to a bright pink. A significant increase in fluorescence signal intensity relative to controls was observed (see Figure 4).

6. Preparation of coumarin-fluorescein cassette

6.1 Preparation of 4-(2-hvdroxymethyl-5-nitro-benzoyl)-DiDerazine-1 - carboxylic acid, tert-butyl ester

Aluminium chloride (447mg) was suspended in anhydrous dichloromethane under an atmosphere of dry nitrogen. A solution of N-Boc- piperazine (1.25g) in dichloromethane (5ml) was added drop wise and the mixture stirred at room temperature for a further 6hrs. A solution of 6- nitrophthalide (600mg) in dichloromethane (10ml) was then added drop wise. The mixture was stirred for a further 2 hrs and then allowed to stand overnight and then quenched by the addition of 1 M hydrochloric acid (5ml) followed by dichloromethane (200ml). The organic phase was washed with water (3x200ml), brine (200ml) and water dried over MgSO₄ and concentrated in vacuo. The residue was purified by silica flash column chromatography (hexane: ethyl acetate (1 :1 ) to ethyl acetate gradient) to give the desired product as a sticky white solid (548mg).

6.2 Preparation of fluorescein intermediate

A solution of disuccinimidylcarbonate (13.3mg) in dry DMF (1 ml) was added to a mixture of the above nitrobenzyl alcohol (18.5mg) and activated powdered molecular sieves in dry DMF (1 ml) followed by triethylamine (21 μl). The resultant mixture was stirred at room temperature for 5hrs under an atmosphere of dry nitrogen. A solution of 5-aminomethylfluorescein hydrochloride (20mg) and triethylamine (42μl) in dry DMF (2ml) was then added and the mixture stirred at room temperature for 3hrs and then allowed to stand overnight. The mixture was then filtered, washed with DMF and concentrated in vacuo. The residue was redissolved in 95% TFA (aq) (10ml), stirred at room temperature for 2hrs and concentrated in vacuo. Purification by reverse phase HPLC (Vydac protein:peptide C18 column; 0.1 % TFA H₂O to 0.1 % TFA MeCN gradient; 10 ml/min) followed by trituration with ethyl acetate afforded the desired fluorescein conjugate as a yellow solid (22mg).

6.3 Preparation of fluorescein-coumarin cassette

To a solution of the above fluorescein conjugate (11 mg), hydroxybenztriazole (1 mg), dimethylaminopyridine (1mg) and 7- diethylaminocoumarin-3-carboxylic acid (5mg) in dry DMF (2ml) was added activated powdered molecular sieves. The resultant mixture was stirred at room temperature for 4hrs and then allowed to stand overnight. The mixture was then filtered, washed with DMF and the filtrate concentrated in vacuo. The residue was then purified in two stages by reverse phase HPLC (Vydac protein:peptide C18 column; 0.1% TFA H₂O to 0.1% TFA MeCN gradient; 10 ml/min) followed by flash silica chromatography (ethyl acetate to 40% ethanol:ethyl acetate gradient). Final trituration with diethyl ether afforded the desired compound as a pale yellow solid (1mg).

Ettan LCMS: C₄₈H₄I N₅Oi₃ requires 895. Found MH⁺ = 896.

7. Evaluation of the fluorescein-coumarin cassette as a reagent for detecting nitroreductase activity

An aliquot of NTR enzyme (40μl of 5.5ng/ml PBS solution) was added to a mixture of NADH (200μl of a 5mg in 640μl PBS solution) and dye substrate (4μl of a 1mM DMSO solution). Reaction progress was monitored by HPLC analysis (Agilient HP1100) equipped with a fluorescence detector (excitation 488nm, emission 530nm). A significant increase in fluorescence signal intensity was observed (see Figure 5).

8. Preparation of Cv3-Cv5 FRET cassette

8.1 Preparation of intermediate

4-(2-Hydroxymethyl-5-nitro-benzoyl)-piperazine-1 -carboxylic acid tert- butyl ester (53mg) was dissolved in anhydrous DMF (3ml) and triethylamine (10OuI) and activated molecular sieves added. The reaction mixture was stirred for 20 minutes after which time N,N'-disuccinimidyl carbonate (DSC) (37mg) was added. After stirring for 4 hours the Cy3™ amine (100mg) was added (as a solution in 3 ml DMF containing 10OuI triethylamine). The reaction was stirred for 3 hours and then left to stand overnight. The desired product was obtained W

30

by preparative HPLC. This material was then treated with 95% TFA (5ml) and stirred for 2 hours to give 49mg of the desired compound.

8.2 Preparation of Cv3-Cv5 cassette

A solution of the carbocyanine prepared in 8.1 above (20mg) and dimethylaminopyridine (5mg) in dry DMF (3ml) was added to a mixture of the Cy5™ carboxylic acid (14mg), hydroxybenztriazole (5mg) and activated powdered molecular sieves in dry DMF (2ml). The mixture was stirred under an atmosphere of dry nitrogen for 20 min and diisopropylcarbodiimide (60ml) was added. After 3hrs the mixture was filtered and the filtrate added to diethyl ether (200ml). The precipitate, thus formed, was collected and purified by reverse phase HPLC (Vydac C18 proetin:peptide; 0.1% TFA H₂O 0.1% TFA MeCN gradient). The main component was collected and triturated with ethyl acetate to afford the desired conjugate (6.3mg).

LCMS: C₇₄H₈SN₈O₁₈S₄ requires 1504. Found M/2 = 752

8.3 Evaluation of the Cv3-Cv5 cassette as a reagent for detecting nitroreductase activity

An aliquot of NTR enzyme (40μl of 5.5ng/ml PBS solution) was added to a mixture of NADH (200μl of a 5mg in 640μl PBS solution) and Cy3-Cy5 dye cassette (4μl of a 1mg/ml DMSO solution). Fluorescence intensity was changes were monitored on a Perkin-Elmer LS55B fluorometer (Excitation 540nm, slit width 10nm; emission 562nm (Cy3) and 670nm (Cy5), slit width 10nm). The fluorescence intensity signal measured at 562nm showed an increase over time, and the fluorescence intensity signal measured at 670nm showed a decrease over time. 9. A comparative study of CvtoCvδ and fluorescein 4-nitrobenzγl ester as substrates in a nitroreductase gene reporter assay

A reporter construct containing four repeats of the NF-κB response ejement upstream of the NTR gene was constructed in plasmid pDC511 (Admax™). This plasmid was co-transfected with the Ad5 genomic DNA into helper cells, HEK293, and recombinant replication incompetent Adenovirus rescued. The individual isolated plaques were screened for correct orientation and sequence prior to amplification in HEK293 cells and subsequent use in cellular assays.

HeLa cells were subcultured twenty-four hours prior to viral transduction and maintained at 37⁰C in a humidified atmosphere of 5% CO₂ in Dulbecco's Modified Eagles medium containing 10% foetal calf serum + 2mM L-glutamine. After the overnight incubation, the cells were detached with trypsin, pooled to produce a suspension of cells and the concentration determined. The HeLa cell suspension was mixed with virus at a predetermined multiplicity of infection (MOI) in a sufficient minimal volume of complete medium to cover the base of a tissue culture flask; typically 15ml for 1-2 x 10⁶ cells in a T75cm² Costar flask. Transduction of HeLa cells was performed at 37⁰C in a humidified atmosphere of 5% CO₂.

The following day the medium was removed from each flask and the transduced cell monolayers rinsed once with 5-1OmI PBS. The cells were detached with trypsin and pooled to produce a suspension of transduced cells; the concentration of the cell suspension was determined and adjusted to 7.5 x 10⁴ cells per ml. 200μl of this cell suspension was dispensed into each well of a 96-well microtitre plate; ~ 1.5 x 10⁴ cells per well. All plates were incubated overnight at 37⁰C in a humidified atmosphere of 5% CO₂. Prior to the assay the overnight culture medium was replaced with 200μl PBS. The PBS was removed and replaced with either 90μl 30ng/ml TNFα in serum free Dulbecco's Modified Eagles medium or 90μl serum free Dulbecco's Modified Eagles medium to the control wells. Plates were returned to the incubator at 37⁰C in an atmosphere of 5% CCMor 2 hours. After this time, 10μl of 10μM solutions either CytoCyδ (shown as compound 3 below) fluorescein 4-nitrobenzyl ester were dispensed individually into replicate wells and plates returned to 37⁰C in a humidified atmosphere of 5% CO2. The fluorescence signal was monitored over time by means of a Tecan "Ultra" fluorimeter using excitation and emission filters designed for Cy5 and fluorescein.

Table 3a

Table 3a shows the fluorescent intensity readings for the CytoCyδ control experiment (i.e. in the absence of TNFα) with respect to time (these experiments were conducted in replicates of 6). Table 3b

Table 3b shows the fluorescent intensity readings for the Cytocyδ stimulated experiment (i.e. in the presence of TNFα) with respect to time (these experiments were conducted in replicates of 6).

Table 3c

Table 3c shows the fluorescent intensity readings for the fluorescein 4-nitro benzyl ester control experiment (i.e. in the absence of TNFα) with respect to time (these experiments were conducted in replicates of 6).

Table 3d

Table 3d shows the fluorescent intensity readings for the fluorescein 4-nitro benzyl ester stimulated experiment (i.e. in the presence of TNFα) with respect to time (these experiments were conducted in replicates of 6).

Figures 7-9 compare the performance of CytoCyδ and fluorescein A- nitrobenzyl ester in a live cell gene reporter assay. These data show that a difference between the control cells and stimulated cells can be detected with a shorter incubation period for the fluorescein 4-nitrobenzyl ester compared with CytoCyδ. At 1 hour post addition of the probes the fluorescein 4-nitrobenzyl ester produced a signal of 333 RFUs in the control samples versus 1633 RFUs for the treated samples. This compares to no observable difference between the control and treated samples incubated with the CytoCyδ. Although, both probes generate very similar signal windows at their optimum incubation time the observable fold induction is far greater at all time points for the fluorescein 4-nitrobenzyl ester, 3.9-9.5, compared to CytoCyδ, 0.98-1.2 (figure 9). The reduction in the fold induction observed for CytoCyδ is a consequence of the high endogenous background signal. In addition the performance of the assay as assessed by signal to noise ratios, has confirmed the fluorescein A- nitrobenzyl ester, signahnoise ratio 9-32, outperformed the CytoCyδ probe, signal:noise ratio 1-3 (Figure 9).

The reduction in background signal and consequently the higher fold induction that was observed with the fluorescein 4-nitrobenzyl ester probe provides a sensitive detection system. This will be important when trying to detect the activity of weak promoters and transcriptional control units. Detection of small changes should be easier to detect with the fluorescein 4- nitrobenzyl ester probe.

Claims

1. A method for determining nitroreductase activity comprising: a) combining a substrate comprising a compound of formula (I):

j) (CH=CH)_m— CH₂ L₁-J-FLUOR

(I) wherein groups R^a and R^b are attached to atoms of the Z ring system;

Z represents the a chain of linked atoms necessary to complete an aromatic or heteroaromatic ring system having five or six atoms selected from carbon atoms and optionally no more than two atoms selected from oxygen, nitrogen and sulphur; FLUOR is a fluorescent dye selected from xanthene, coumarin and oxazine dyes; L₁ is a bond or is an atom or a chain of 2 to 10 covalently linked atoms selected from the group consisting of carbon, nitrogen, oxygen and sulphur atoms; each R^a is hydrogen or may be selected from electron donating and withdrawing groups, and p is 0 or an integer from 1 to 3; R^b is hydrogen or is the group -L₂-W, where L₂ is a linker chain containing from 1- 20 linked atoms and W is a fluorescent dye moiety or a quenching group; m is 0 or 1 ; and r is 1 or 2; with at least a portion of a sample containing or suspected to contain nitroreductase under conditions to cause cleavage of FLUOR from said substrate; and b) detecting a change in an optical property of said sample following the combining step a); wherein said change is a measure of the amount of nitroreductase activity.

2. A method according to claim 1 , wherein the sample is a tissue sample.

3. A method according to claim 1 , wherein the sample is a cell extract.

4. A method according to claim 1 , wherein the sample comprises at least one cell.

5. A method according to claim 1 , wherein the substrate is, or is rendered permeable to cells.

6. A method of screening for a test agent whose effect upon the activity of a nitroreductase is to be determined, said method comprising the steps of:

(a) performing the method according to any of claims 1 to 5 in the presence and in the absence of said agent; and

(b) determining the activity of the enzyme in the presence and in the absence of said agent; wherein a difference between the activity of said enzyme in the presence and in the absence of said agent is indicative of the effect of said test agent on the activity of said enzyme.

7. A method of screening for a test agent whose effect upon the activity of a nitroreductase is to be determined, said method comprising the steps of:

(a) performing the method according to any of claims 1 to 5 in the presence of said agent; and

(b) comparing the value of the activity of the enzyme with a control value for the enzyme activity in the absence of the test agent.

8. A method according to claim 7, wherein said control value is stored electronically in a database or other electronic format.

9. A method for detecting nitroreductase gene expression, said method comprising the steps of: a) providing a host cell wherein said host cell has been transfected with a nucleic acid molecule comprising expression control sequences operably linked to a sequence encoding a nitroreductase; b) contacting said host cell with a substrate under conditions to promote nitroreductase activity and wherein said substrate comprises a compound of formula (I):

J) (CH=CH)_m— CH₂ — Lf-f-FLUOR

(I) wherein groups R^a and R^b are attached to atoms of the Z ring system; Z represents the a chain of linked atoms necessary to complete an aromatic or heteroaromatic ring system having five or six atoms selected from carbon atoms and optionally no more than two atoms selected from oxygen, nitrogen and sulphur; FLUOR is a fluorescent dye selected from xanthene, coumarin and oxazine dyes; Li is a bond or is an atom or a chain of 2 to 10 covalently linked atoms selected from the group consisting of carbon, nitrogen, oxygen and sulphur atoms; each R^a is hydrogen or may be selected from electron donating and withdrawing groups, and p is 0 or an integer from 1 to 3; R^b is hydrogen or is the group -L₂-W, where L₂ is a linker chain containing from 1- 20 linked atoms and W is a fluorescent dye moiety or a quenching group; m is 0 or 1 ; and r is 1 or 2; and c) detecting a change in an optical property upon cleavage of said FLUOR from said substrate; wherein said change is a measure of the amount of nitroreductase gene expression.

10. A method according to claim 9, wherein the substrate is or is rendered permeable to cells.

11. A method according to any of claims 1 to 10, wherein said optical property is fluorescence emission intensity of said FLUOR.

12. A method according to any of claims 1 to 11 , wherein Z is selected from phenyl, naphthyl, imidazolyl, oxazolyl and thiazolyl moieties.

13. A method according to claim 12, wherein Z is a phenyl or an imidazolyl ring system.

14. A method according to any of claims 1 to 13, wherein R^a is selected from hydrogen, cyano, halogen, hydroxyl, Ci-C₄ alkyl, -NO₂, -NHCO₂R^d, -CO₂H, -CO₂R^d, -SH, Ci-C₄ alkylamino, and C₁-C4 alkoxyl; where R^d is C₁-C₄ alkyl.

15. A method according to any of claims 1 to 13 wherein R^a is selected from Ci-C₄ alkyl, C1-C4 alkoxyl and C₁-C₄ alkylamino.

16. A method according to any of claims 1 to 15, wherein said substrate is a compound of formula:

wherein L₁ and FLUOR are defined as in claim 1.

17. A method according to any of claims 1 to 16, wherein FLUOR is a xanthene dye.

18. A method according to any of claims 1 to 16, wherein FLUOR is a coumarin dye.

19. A method according to any of claims 1 to 16, wherein FLUOR is an oxazine dye.

20. A method according to any of claims 1 to 16, wherein R^b is the group -L₂-W, where L₂ and W are hereinbefore defined.

21. A method according to claim 20, wherein W is a dye moiety selected from coumarin dyes, benzocoumarin dyes, phenoxazine dyes, xanthene dyes, cyanine dyes and derivatives of the bis-pyrromethine boron difluoride dyes.

22. A method according to claim 20, wherein W is a quenching group selected from 2,4-dinitrophenyl (DNP) and 4-(4-dimethylaminophenyl)- azobenzoic acid (DABCYL). selected from groups such as 2,4-dinitrophenyl (DNP) and 4-(4-dimethylaminophenyl)-azobenzoic acid (DABCYL), Malachite green, QSY 7, QSY 21 , QSY 35, methyl red, methyl orange and Black Hole Quencher™.

23. A method according to any of claims 1 to 16, wherein said substrate is a compound of formula:

wherein X is OR¹² or NR¹²R¹³ and Y is O or N⁺R¹²R¹³; R¹, R², R³, R⁴, R⁵, R⁶ and R⁷ are selected from hydrogen, halogen, sulphonic acid, sulphonate, cyano, Ci-C₄ alkyl, Ci-C₄ perfluoro and Ci-C₄ alkoxyl; at least one of groups R⁸, R⁹, R¹⁰, R¹¹, R¹² and R¹³ is the group:

wherein groups R^a, R^b, Li, Z, p and m are hereinbefore defined; remaining groups R⁸, R⁹ and R¹⁰ are independently selected from hydrogen halogen, carboxyl, sulphonic acid, sulphonate, cyano, Ci-C₄ alkyl, Ci-C₄ perfluoro and Ci-C₄ alkoxyl; remaining groups R¹¹, R¹², and R¹³ are independently selected from hydrogen and Ci-C₄ alkyl or when Y is N⁺R¹²R¹³, R¹² taken in combination with R¹ and R¹³ taken in combination with R², together form a fused tricyclic ring system each ring containing 6 atoms; and remaining group R¹¹ is hydrogen or Ci-C₄ alkyl, preferably methyl or ethyl.

24. A method according to any of claims 1 to 16, wherein said substrate is a compound of formula:

wherein X is OR 1¹2' or

at least one of groups R >12 and R ,13 is the group:

wherein groups R^a, R^b, Li, Z, p and m are hereinbefore defined; remaining group R >12, or R »13 is selected from hydrogen and Ci-C₄ alkyl; and

R ,1¹4* is selected from Ci-C₄ alkyl, CF₃, CO₂H and CH₂CO₂H.

25. A method according to any of claims 1 to 16, wherein said substrate is a compound of formula:

wherein X is OR >1"2 or

and Y is O or

wherein at least one group R¹² or R¹³ is the group:

wherein groups R^a, R^b, Li, Z, p and m are hereinbefore defined; and remaining groups R¹² and R¹³ are selected from hydrogen and Ci-C₄ alkyl.

26. A kit for a reporter system comprising: i) means for expressing a nitroreductase enzyme; and ii) a compound of formula (I).

27. A kit according to claim 26, wherein said means comprises a nucleic acid comprising expression control sequences operably linked to a sequence encoding a nitroreductase and being optionally linked to a target gene of interest.

28. A compound of formula:

-0-C(O)-,

-0-C(O)-O-,

-0-C(O)-NH-, and

-0-C(O)-NH-CH₂- ; remaining groups R⁸, R⁹ and R¹⁰ are independently selected from hydrogen halogen, carboxyl, sulphonic acid, sulphonate, cyano, C1-C₄ alkyl, C₁-C₄ perfluoro and C₁-C₄ alkoxyl; remaining groups R¹¹, R¹², and R¹³ are independently selected from hydrogen and Ci-C₄ alkyl or when Y is N⁺R¹²R¹³, R¹² taken in combination with R¹ and R¹³ taken in combination with R², together form a fused tricyclic ring system each ring containing 6 atoms; and remaining group R¹¹ is hydrogen or C1-C₄ alkyl, preferably methyl or ethyl.

29. A compound of formula:

wherein groups R^a, R^b, Z, p and m are hereinbefore defined; Li is a chain of covalently linked atoms selected from:

-0-C(OK -0-C(O)-O-, -0-C(O)-NH-, and -0-C(O)-NH-CH₂- ; remaining group R¹²or R¹³ is selected from hydrogen and C₁-C₄ alkyl; and R¹⁴ is selected from C₁-C₄ alkyl, CF₃, CO₂H and CH₂CO₂H.

30. A compound of formula:

O₂h

_(Ra_)p_i-_ Z J) (CH=CH)_m-CH₂— L₁

≠^^"—^"' wherein groups R^a, R^b, Li, Z, p and m are hereinbefore defined; and remaining groups R¹² and R¹³ are selected from hydrogen and Ci-C₄ alkyl.

31. A compound according to any of claims 28 to 30, wherein Z is a phenyl or an imidazolyl ring system.

32. A compound according to any of claims 28 to 30, wherein the group:

is selected from:

wherein R^a, R^b, R^c, Li and p are hereinbefore defined and m is 0.

33. A compound according to any of claims 28 to 32, wherein said compound is, or is rendered, permeable to cells.

34. A compound selected from:

35. Use of a reagent for preparing a compound of formula (I): J) (CH=CH)_m-CH₂ — L₁-J-FLUOR

(D wherein groups R^a and R^b are attached to atoms of the Z ring system; Z represents the a chain of linked atoms necessary to complete an aromatic or heteroaromatic ring system having five or six atoms selected from carbon atoms and optionally no more than two atoms selected from oxygen, nitrogen and sulphur; FLUOR is a fluorescent dye; Li is a bond or is an atom or a chain of 2 to 10 covalently linked atoms selected from the group consisting of carbon, nitrogen, oxygen and sulphur atoms; each R^a is hydrogen or may be selected from electron donating and withdrawing groups, and p is 0 or an integer from 1 to 3; R^b is hydrogen or is the group -L₂-W, where L₂ is a linker chain containing from 1-20 linked atoms and W is a fluorescent dye moiety or a quenching group; m is 0 or 1 and r is 1 or 2; and wherein said reagent is a compound of formula (II):

(H) wherein groups R^a, R^b, Z, Li, p and m are hereinbefore defined and Q is a reactive group.

36. Use according to claim 35, wherein Q is a group chosen so as to be reactive with a complementary functional group of FLUOR.

37. Use according to claim 35, wherein Q is a leaving group selected from halogen, Ci-C₄ alkoxy, O-mesylate and O-triflate.

38. Use according to claim 35, wherein Z is a phenyl or an imidazolyl ring system.

39. Use according to any of claims 35 to 38, wherein FLUOR is a xanthene dye.

40 Use according to any of claims 35 to 38, wherein FLUOR is a coumarin dye.

41. Use according to any of claims 35 to 38, wherein FLUOR is an oxazine dye.

42. Use according to any of claims 35 to 38, wherein FLUOR is a cyanine dye.