Patent Publication Number: US-2004053270-A1

Title: RNA binding compounds and uses thereof

Description:
RELATED APPLICATIONS  
     [0001] This application is a continuation of PCT/GB01/03611, filed Aug. 13, 2001, which claims the benefit of GB Application No. 0020083.2, filed Aug. 15, 2000, the entirety of which is hereby incorporated by reference. 
    
    
     
       TECHNICAL FIELD  
       [0002] This invention is in the field of assays for compounds which interact with RNA. More particularly, it is in concerned with the identification of improved antibiotics.  
       BACKGROUND ART  
       [0003] In most biological systems, the maturation, transport, stability and expression of RNA is closely regulated by the interactions between highly conserved regulatory RNA sequences and proteins. In many circumstances it is desirable to develop drugs that bind RNA at sites of regulatory protein binding and act as competitive inhibitors of the RNA-protein interaction. These types of drugs have potential application in a wide range of diseases including viral, bacterial and fungal infections and chronic diseases such as cancer and autoimmune disease.  
       [0004] To identify RNA-binding compounds, methods involving fluorescence have been reported.  
       [0005] Reference 1 discloses methods in which a compound and a RNA are labelled with fluorescent donor and acceptor groups. Interaction between the compound and RNA allows the donor and acceptor groups to interact, resulting in a detectable change (quenching). Reference 2 discloses methods in which aminoglycosides are fluorescently labelled in order to monitor their binding to RNA molecules. Both references point towards reducing the size of the RNA towards a “minimal motif” for use in the assay.  
       DISCLOSURE OF THE INVENTION  
       [0006] The invention is based on the finding that reducing the size of RNA does not necessarily give the best results. In particular, it has been found that small RNA molecules of the type disclosed in reference 2 may not bind antibiotics, whereas larger RNA molecules or complexes retain antibiotic-binding activity. This suggests that tertiary interactions are important for the formation of an antibiotic-RNA complex.  
       [0007] The invention provides a method for determining whether a test compound binds to a large RNA target, the method comprising the steps of:  
       [0008] (a) contacting the test compound with a pair of indicator molecules comprising (i) the large RNA target; and (ii) a fluorescent reporter molecule, wherein the reporter molecule is an oxazolidinone or an aminoglycoside; and  
       [0009] (b) measuring the fluorescence of reporter molecule in the presence of the test compound and comparing this value to the fluorescence of the reporter in the absence of the test compound.  
       [0010] In contrast to the methods disclosed in reference 1, the fluorescent group on the reporter does not interact with a fluorescent group on the RNA target.  
       [0011] The Large RNA Target  
       [0012] Contrary to the trends in the prior art, the method of the present invention utilises large RNA molecules.  
       [0013] The RNA target will comprise more than 250 nucleotides, typically more than 350 nucleotides, and often more than 500 nucleotides.  
       [0014] The RNA target will be able to fold to adopt a complex structure. In particular, it may contain up to N base pairs, where N=(total number of nucleotides in RNA)/2 e.g. at least 0.5N base pairs, preferably at least 0.7N base pairs, more preferably at least 0.8N base pairs, and most preferably 0.9N base pairs.  
       [0015] The RNA target will preferably comprise at least one of the following secondary structure motifs (i.e. at least one discontinuity in normal base-paired double-helical RNA): bulged (non base-paired) and extra-helical bases; internal loops; helical junctions; G-quartets; and pseudoknots. The RNA target may comprise inter-domain (inter-helical or inter-secondary structure) molecular interactions, which may include the ligand binding site.  
       [0016] It may also comprise protein in the form of a RNP.  
       [0017] Examples of preferred RNA targets include viral genomes, RNAs comprising an IRES, nucleoprotein complexes (e.g. RNPs, SNRPs), mRNAs, rRNAs (e.g. complete rRNAs such as 23S rRNA, 16S rRNA etc.), ribosomal subunits, and whole ribosomes.  
       [0018] The RNA target may comprise one or more RNA strands, and it may comprise natural or synthetic RNA.  
       [0019] The Fluorescent Reporter p RNA-binding molecules typically contain functional groups (e.g. primary and secondary amine, hydroxyl, nitro, carbonyl etc.) which can be derivatised using fluorescent dyes to prepare reporters. Thus the reporter is prepared by labeling a compound which (a) binds to RNA e.g. an antibiotic and (b) has an atom to which a fluorophore can be covalently attached. The labelled compound can undergo a measurable change when it binds to RNA, and is thus useful in binding and displacement assays.  
       [0020] The fluorescent reporter used in the methods of the invention is an oxazolidinone or an aminoglycoside, to which is attached a suitable fluorophore. The fluorophore may be any fluorophore which does not interfere with the ability of the oxazolidinone or aminoglycoside to interact with the RNA target. A preferred fluorophore is TAMRA.  
       [0021] Other useful fluorophores include, but are not limited to: Texas Red™ (TR), Lissamine™ rhodamine B, Oregon Green™ 488 (2′,7′-difluorofluorescein), carboxyrhodol and carboxyrhodamine, Oregon Green™ 500, 6-JOE (6-carboxy-4′,5′-dichloro-2′,7′-dimethyoxyfluorescein, eosin F3S (6-carboxymethylthio-2′,4′,5′,7′-tetrabromotrifluorofluorescein), cascade blue™ (CB), aminomethylcoumarin (AMC), pyrenes, dansyl chloride (5-dimethylaminonaphthalene-1-sulfonyl chloride) and other naphthalenes, PyMPO, ITC (1-(3-isothiocyanatophenyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridinium bromide).  
       [0022] Oxazolidinones are antibiotics which contain a substituted 2-oxazolidinone group:  
                 
 
       [0023] It will be appreciated that various substitutions can be made in this ring structure without affecting antibiotic activity. In particular, the NH group can be substituted with various groups (typically aromatic) and the CH 2  adjacent to the —O— group can be substituted (e.g. with —CH 2 NH—COCH 3 ) [see, for example, references 3, 4, 5, 6, 7, 8, 9 etc.] 
       [0024] According to a preferred embodiment, the oxazolidinone employed in the present invention has the formula  
                 
 
       [0025] wherein R 1  to R 5  are each independently selected from a σ bonding substituent.  
       [0026] Preferably R 1  to R 5  are independently selected from halogen, hydrogen, C 1-12  alkyl, C 2-12  alkenyl, C 3-12  aryl, C 4-18  aralkyl.  
       [0027] Preferably R 1  is an optionally substituted C 3-12  aryl group. More preferably R 1  is a substituted phenyl group. More preferably, R 1  is a phenyl group substituted with 1 to 3 substituents, preferably 1 or 2 substituents, selected from halogen and heterocyclic groups. The heterocyclic groups may be further substituted.  
       [0028] Preferably R 2 , R 3  and R 4  are hydrogen.  
       [0029] Preferably R 4  and R 5  are independently selected from hydrogen and substituted C 1-12  alkyl. More preferably R 4  is hydrogen and R 5  is substituted C 1-12  alkyl. More preferably R 5  is an acetamidomethyl group. Oxazolidinone antibiotics are reviewed in References 25 and 26.  
       [0030] Oxazolidinones may have aldehyde groups which can be labelled with a fluorophore. Labelled oxazolidinones are preferred reporters for use in the invention. The label is preferably TAMRA, which may be attached as shown in FIG. 1. Preferred large RNA targets for use with oxazolidinone reporters comprise the E-site RNA (FIG. 3) or the L1 (e.g. a rRNA).  
       [0031] Preferred oxazolidinones are those which interact at the ribosome E (‘exit’) site [10].  
       [0032] A preferred oxazolidinone is “DuPont 721”, which binds to the 23S subunit of the ribosome at the nucleotides and ribosomal proteins that constitute the E site for tRNA binding, including nucleotides 2113, 2114, 2118, 2119 and 2153. There are also interactions with nucleotide 864 in the 16S subunit [3].  
       [0033] As used herein, the term “alkyl” means an optionally substituted branched or unbranched, cyclic or acyclic, hydrocarbyl radical. Where acyclic, the alkyl group is preferably a C 1-12 , more preferably C 1-4  chain. Where cyclic, the alkyl group is preferably a C 3-12 , more preferably C 5-10  and more preferably comprises a C 5 , C 6  or C 7  ring.  
       [0034] As used herein, the term “alkenyl” means an optionally substituted branched or unbranched, cyclic or acyclic, hydrocarbyl radical comprising at least one double bond. Where acyclic, the alkenyl group is preferably a C 1-12 , more preferably C 1-4  chain. Where cyclic, the alkenyl group is preferably a C 3-12 , more preferably C 5-10  and more preferably comprises a C 5 , C 6  or C 7  ring.  
       [0035] As used herein, the term “aryl” means an optionally substituted C 3-12  aromatic group, such as phenyl or naphthyl, or a heteroaromatic group containing one or more, preferably one, heteroatom, such as pyridyl, pyrrolyl, furanyl, thienyl.  
       [0036] As used herein, the term “aralkyl” means an optionally substituted branched or unbranched cyclic or acylic C 4-18  group comprising an alkyl group and an aryl group (for example, benzyl). An aralkyl group may be bonded via the alkyl or aryl group.  
       [0037] The alkyl, alkenyl, aryl, aralkyl and heterocyclic groups may be substituted or unsubstituted. Where substituted, there are preferably one to three substituents, more preferably one substituent. Substituents may include halogen atoms and halogen containing groups such as haloalkyl (e.g. trifluoromethyl); oxygen containing groups such as alcohols (e.g. hydroxy, hydroxyalkyl, aryl(hydroxy)alkyl), ethers (e.g. alkoxy, alkoxyalkyl, aryloxyalkyl), aldehydes (e.g. carboxaldehyde), ketones (e.g. alkylcarbonyl, alkylcarbonylalkyl, arylcarbonyl, arylalkylcarbonyl, arylcarbonylalkyl), acids (e.g. carboxy, carboxyalkyl), acid derivatives such as esters (e.g. alkoxycarbonyl, alkoxycarbonylalkyl, alkycarbonylyoxy, alkycarbonylyoxyalkyl) and amides (e.g. aminocarbonyl, mono- or dialkylaminocarbonyl, aminocarbonylalkyl, mono- or dialkylaminocarbonylalkyl, arylaminocarbonyl); and carbamates (e.g. alkoxycarbonylamino, aryloxycarbonylamino, aminocarbonyloxy, mono- or dialkylaminocarbonyloxy, arylaminocarbonyloxy), and ureas (e.g. mono- or dialkylaminocarbonylamino or arylaminocarbonylamino); nitrogen containing groups such as amines (e.g. amino, mono- or dialkylamino, aminoalkyl, mono- or dialkylaminoalkyl), azides, nitriles (e.g. cyano, cyanoalkyl), nitro; sulfur containing groups such as thiols, thioethers, sulfoxides, and sulfones (e.g. alkylthio, alkylsulfinyl, alkylsulfonyl, alkylthioalkyl, alkylsulfonylalkyl, alkylsulfonylalkyl, arylthio, arylsulfinyl, arylsulfonyl, arylthioalkyl, arylsulfinylalkyl, arylsulfonylalkyl). The alkyl, alkenyl, aryl and aralkyl groups may also be substituted with heterocyclic groups containing one or more, preferably one, heteroatom (e.g. thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, oxadiazolyl, thiadiazolyl, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, tetrahydrofuranyl, pyranyl, pyronyl, pyridyl, pyrazinyl, pyridazinyl, piperidyl, piperazinyl, morpholinyl, thianaphthyl, benzofuranyl, isobenzofuranyl, indolyl, oxyindolyl, isoindolyl, indazolyl, indolinyl, 7-azaindolyl, benzopyranyl, coumarinyl, isocoumarinyl, quinolinyl, isoquinolinyl, naphthridinyl, cinnolinyl, quinazolinyl, pyridopyridyl, benzoxazinyl, quinoxalinyl, chromenyl, chromanyl, isochromanyl, phthalazinyl and carbolinyl).  
       [0038] As used herein, the term “halogen” means a fluorine, chlorine, bromine or iodine radical, preferably a fluorine or chlorine radical. It will be appreciated that the compounds of the present invention may exist in a number of diastereomeric and enantiomeric forms. The present invention encompasses pure diastereomers and enantiomers as well as mixtures (including racemic mixtures) of diastereomers and enantiomers.  
       [0039] Aminoglycosides are broad-spectrum antibiotics that contain an aminodeoxysugar, an amino- or guanidino-substituted inositol ring, and one or more residues of other sugars. Preferably, the aminoglycosides contain one or more aminocyclitol rings (hexose nucleus), amino or guanidino-substituted inositol rings and may be attached to aminodeoxysugars or other sugars by glycosidic bonds. The structure of various aminoglycosides are shown in reference 11. Preferred classes and individual compounds are set out in the following table.  
                                                                                       in                               Acting   Order   clinical   Vet           Class   Antibiotic   Synonyms   Target   on   information   use   use   Ref.                  4,6-disubstituted   Amikacin       A site       Sigma   +       J Infect Dis 1998 Jun;       deoxystreptamine                   A3650           177(6): 1573-81       core       Aminoglycoside   Apramycin   Nebramycin II   A site       Sigma   −   +   EMBO J 1991 Oct;                           A2024           10(10): 3099-103       4,6-disubstituted   Kanamycin B   Bekanamycin   A site       Sigma           Biochemistry 1998 Jan 13;       deoxystreptamine                   B5264           37(2): 656-63       core       Aminoglycoside   Bluensomycin       ?       ?           RNA 1998 Jan; 4(1): 112-       (streptomycin                               23       type)       4,5-disubstituted   Butirosin A       deoxystreptamine       core       Aminoglycoside   Fortimycin       4,6-disubstituted   Gl 8               Sigma           Biochemistry 1984 Mar       deoxystreptamine                   G5013           27; 23(7): 1462.7       core       4,6-disubstituted   Gentamicin   Gentamycin   A site       Sigma   +       EMBO J 1998 Nov 16; I       deoxystreptamine           (L6)       G1264           7(22): 6437-48       core                   (G6896)       Aminoglycoside   Hygromycin B                           Nature 1987 June 4-10;                                       327(6121): 389-94       Aminoglycoside   Istamycin       A site                   Mol Gen Genet 1984;                                       197(1): 24-9       4,6-disubstituted   Kanamycin A       A site       Sigma   +       J Mol Biol 1987 Feb 20;       deoxystreptamine                   K0879           193(4): 661-71       core       Aminoglycoside   Kasugamycin       P site   pro   Sigma-           EMBO J 1991 Oct;                           Aldrich rare           10(I0): 3099-103                           chemicals                           78, 711-6       Aminoglycoside   Lividomycin A       (Neomycin       group)       Aminoglycoside   Myomycin       ?       ?           EMBO J 1991 Oct;                                       10(10): 3099-103       Aminoglycoside   Neamin       A site                   EMBO J 1991 Oct;       (Neomycin                               10(10): 3099-103       group)       4,5-disubstituted   Neomycin   Fradiomycin   A site       Sigma   +       J Mol Biol 1999 Feb 12;       deoxystreptamine                   N1876           286(1): 33-43       core       4,6-disubstituted   Netilmicin       ?       Sigma   +       J Antimicrob Chemother       deoxystreptamine                   N0755           1984 Sep; 14(3): 231-41       core       4,5-disubstituted   Paromomycin       A site       Calbiochem           J Mol Biol 1998 Mar 27;       deoxystreptamine                   512731           277(2): 333-45       core       4,5-disubstituted   Ribostamycin       A site       Sigma           J Mol Biol 1998 Mar 27;       deoxystreptamine                   R2255           277(2): 347-62       core       Aminoglycoside   Sagamicin       ?                   J Antibiot (Tokyo) 1983       (Kanamycin                               Feb; 36(2): 125.30       group)       4,6-disubstituted   Sisomycin       ?       Calbiochem           J Bacteriol 1992 Dcc;       deoxystreptamine                   567205           174(23): 7868-72       core       Aminoglycoside   Sorbistin       ?                   J Antibiot (Tokyo) 1976                                       Nov; 29(11): 1152-62       Aminocylitol   Spectinomycin       S5   pro   Calbiochem   +   +   J Mol Biol 1997 Aug 29;                   binding       567570           271(4): 566-87                   site       Aminoglycoside   Streptomycin   Actinamine   A site,       Caliochem   +       J Mol Biol 1997 Oct 31;       (streptomycin           915       5711           273(3): 586-99       type)           region,                   S12,                   S5?,                   L11?       4,6-disubstituted   Tobramycin   Streptidine   A site       Sigma   +       Nature 1987, 327, 389-       deoxystreptamine                   T4014           394       core       Aminoglycoside   Trospectomycin       ?                   J Chemother 1995 Dcc;                                       7(6): 515-8       4,5-disubstituted   Isepamycin       deoxystreptamine       core       4,5-disubstituted   Kanamycin C       deoxystreptamine       core       4,5-disubstituted   Arbekacin       deoxystreptamine       core                  
 
       [0040] The following aminoglycosides have an amine group which can be labelled with a fluorophore:  
                                                   Class   Antibiotic   Target   RRNA   Nucleotides(s)   Ref                  Aminoglycoside   Amikacin   A site   16S   1408   11       Aminoglycoside   Apramycin   A site   16S   1408 1419 1494   12       Aminoglycoside   Bekanamycin   site   16S   ?   13       Aminoglycoside   Gentamicin   A site (L6)   16S   1408 1419 1494   14       Aminoglycoside   Hygromycin B       16S   1491 1495   15       Aminoglycoside   Kanamycin   A site   16S   1408 1419 1494   16       Aminoglycoside   Kasugamycin       163    794 926   12       Aminoglycoside   Neamin   A site   16S   1408 1419 1494   12       Aminoglycoside   Neomycin   A site   16S   1408 1419 1494   17       Aminoglycoside   Paromomycin   A site   16S   1408 1419 1491 1494   18       Aminoglycoside   Sisomycin               19       Aminoglycoside   Spectinomycin   S5 binding site   16S   1063-1065 1191-1193   20       Aminoglycoside   Tobramycin   A site   163   ?   21                  
 
       [0041] A number of reactions can be used to label amines, including but not limited to the following:  
                                                   Reaction   Product                          dye-isothiocyanates   Thiourea           dye-succinimidyl ester   Carboxamide           dye-sulfonyl chloride   Sulphonamide           dye-aldehyde   Alkylamine                      
 
       [0042] Streptomycin contains an aldehyde group that is appropriate for the introduction of fluorescent dyes:  
                                                   Class   Antibiotic   Target   rRNA   Nucleotides(s)   ref                  Amino-   Streptomycin   A site, 915   16S   523 911-915   22       glycoside       region, S12,               S5?, L11?                  
 
       [0043] A number of reactions can be used to label aldehydes, including but not limited to the following:  
                                                   Reaction   Product                          dye-hydrazides   Hydrazones           dye-semicarbazides   Hydrazones           dye-carbohydrazides   Hydrazones           dye-amines   Alkylamine                      
 
       [0044] Preferred aminoglycosides are those which interact at the ribosome A site. Preferred aminoglycosides for use according to the invention are 4,5-disubstituted deoxystreptamines, such as neomycin and paromomycin, in particular paromomycin. A further preferred aminoglycoside is streptomycin. A further group of preferred aminoglycosides are 4,6 deoxystreptamines, such as tobramycin and gentamycin.  
                                                                                                      R 1                         Neomycin   NH 2             Paramomycin   OH                                                                                                                                                   R 2     R 3     R 4     R 5     R 6                         Tobramycin   NH 2     OH   OH   H   H           Gentamycin   NHCH 3     H   NH 2     CH 3     CH 3                        
 
       [0045] It is surprising that fluorescent labels can be attached to RNA-binding compounds without affecting the ability of these compounds to bind to large RNA molecules because (a) the chemical groups to which labels can be attached are generally the same as those which interact with RNA and (b) even though it may be relatively large (e.g. doubling the size of the initial molecule), the fluorescent label does not interfere with the binding interaction. The reporter will not typically possess intrinsic fluorescence in its unlabelled form.  
       [0046] In contrast to the methods disclosed in reference 1, the fluorescent group on the reporter does not interact with a fluorescent group on the RNA target. The alignment of fluorescent groups in reference 1 implies that the site in the RNA which interacts with the reporter is known whereas, in the present invention, this is not necessary; where this information is known, however, the label will typically be attached at a site remote from the main interacting groups.  
       [0047] It will be appreciated that the methods of the present invention are not aimed at revealing sites within RNA which are bound by the reporter. Conversely, however, they may reveal the sites within the reporter which bind to the RNA.  
       [0048] The Test Compound  
       [0049] The method of the invention may be used to identify compounds capable of binding to any large RNA target, preferably as part of a screening process.  
       [0050] Typical test compounds include, but are not restricted to, peptides, peptoids, proteins, lipids, metals, nucleotides, nucleosides, small organic molecules, antibiotics, polyamines, and combinations and derivatives thereof. Small organic molecules have a molecular weight of more than 50 and less than about 2,500 daltons, and most preferably between about 300 and about 800 daltons. Complex mixtures of substances, such as extracts containing natural products, or the products of mixed combinatorial syntheses, can also be tested and the component that binds to the target RNA can be purified from the mixture in a subsequent step.  
       [0051] Test compounds may be derived from large libraries of synthetic or natural compounds. For instance, synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK) or Aldrich (Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts may be used. Additionally, test compounds may be synthetically produced using combinatorial chemistry either as individual compounds or as mixtures.  
       [0052] Test compounds which can displace reporters based on known antibiotics are useful lead compounds for the development of improved antibiotics.  
       [0053] Indicator Molecules  
       [0054] Contact between the pair of indicator molecules may occur in solution (e.g., a test tube, dish or well of a microtitre plate) or, alternatively, either the reporter molecule or the RNA target molecule may be adhered to a solid support (e.g. an affinity gel, matrix, or column) by covalent or non-covalent linkages using methods known in the art. The support bound RNA target or reporter molecule is then mixed with a solution containing the other compound of the indicator pair.  
       [0055] In some embodiments, a fraction of the reporter molecules and RNA target molecules in the binding reaction can be replaced by unlabelled analogues. The optimal proportions of labelled and unlabelled reporting and RNA target molecules can be determined by titration of the different components and measuring the optimal concentrations.  
       [0056] The RNA and labelled reporter molecules are then mixed with a test compound and the fluorescence in the mixture is measured. If the test compound is able to bind to the region of the target RNA that binds to the reporter, then a fraction of the reporter will be prevented from binding to the RNA target. The proportions of the free reporter, free RNA and complex can be quantitatively determined by comparing the special properties of the complex, partially dissociated complex and the uncomplexed target RNA and reporters. The amount of reporter displacement will be a function of the relative affinity of the test compound for the RNA target compared to the reporter and the relative concentrations of the two molecules in the sample. Preferably, a variety of different concentrations of the molecule to-be-tested are compared to generate a binding curve.  
       [0057] The concentration of compounds binding to RNA targets can be determined with a fluorescence standard curve depicting the fluorescence of the labelled reporter and RNA targets with varying known concentrations of competing unlabelled test compound.  
       [0058] In some embodiments of the invention, the test compound is first mixed with the RNA in order to form a complex in the absence of the labelled reporter, and the reporter is then added. Since the reporter will only be able to bind to the free RNA in the reaction, there will be a reduced amount of complex formed between the reporter and the RNA target compared to the amount of complex formed in the absence of test compound.  
       [0059] In other embodiments, a complex is pre-formed between the RNA and the labelled reporter before addition of the test compound. If the test compound is able to disrupt the complex formed between the RNA and the labelled reporter, or alter the equilibrium binding state by binding to RNA that has dissociated from the reporter, the amount of complex in the reaction will be reduced.  
       [0060] Measurable Changes  
       [0061] The displacement of the fluorescent reporter from the large RNA target can be measured by a number of techniques, including:  
       [0062] fluorescence anisotropy [(I // −I⊥)/(I // +2I⊥), where I //  is the fluorescence intensity viewed through parallel polarisers and I⊥ is the fluorescence intensity viewed through orthogonal polarisers]. This is a particularly useful method when dealing with large RNA targets. In solution, a reporter is generally rotationally free with low anisotropy. When bound to the RNA target, however, they become rotationally constrained and anisotropy is high. Displacement of the reporter is thus associated with a reduction in anisotropy.  
       [0063] fluorescence polarisation [(I // −I⊥)/(I // +I⊥)] 
       [0064] a change in fluorescence intensity or quenching occurring on dissociation. The intensity of many fluorophores depends on the local environment. The change from free solution to the environment of the large RNA target may thus result in a change in fluorescence intensity and/or quenching.  
       [0065] The method includes the step of comparing the fluorescence of reporter molecule in the presence and absence of the test compound. It will be appreciated that the fluorescence of the reporter in the absence of the test compound may have been determined before performing the method, or may be determined during or after the method has been performed. It may be an absolute standard.  
       [0066] Library Screening (Including High Throughput Screens)  
       [0067] The present invention also encompasses high-throughput screening methods for identifying compounds that bind to a RNA target. Preferably, all the biochemical steps for this assay are performed in a single solution in, for instance, a test tube or microtitre plate, and the test compounds are analyzed initially at a single compound concentration. For the purposes of high throughput screening, the experimental conditions are adjusted to achieve a proportion of test compounds identified as “positive” compounds from amongst the total compounds screened. The assay is preferably set to identify compounds with an appreciable affinity towards the RNA target e.g., when 0.1% to 1% of the total test compounds from a large compound library are shown to bind to a given RNA target with a Ki of 10 μM or less (e.g. 1 μM, 100 nM, 10 nM, or less).  
       [0068] Kits Useful According to the Invention  
       [0069] The invention also provides a kit for determining whether a text compound binds to a large RNA target, the kit comprising (i) a large RNA target; and (ii) a fluorescent reporter molecule, wherein the reporter molecule is an oxazolidinone or an aminoglycoside.  
       [0070] Measurements of RNA Binding Compound  
       [0071] The invention may be embodied as a clinical assay or method for determining the presence of an RNA-binding compound in a biological sample such as the serum or tissues of a subject. Many drugs, including RNA-binding compounds such as antibiotics, are routinely assayed for their serum levels when administered to patients to prevent administration of toxic levels of compounds.  
       [0072] The invention thus provides a method for determining the presence in a biological sample of a compound that binds to a large RNA target, the method comprising the steps of (a) contacting the sample with a pair of indicator molecules comprising (i) the large RNA target; and (ii) a fluorescent reporter molecules; wherein the reporter molecule is an oxazolidinone or an aminoglycoside; and (b) measuring the fluorescence of the reporter molecule in the presence of the sample and comparing this value to the fluorescence of the reporter in the absence of the test compound.  
       [0073] The invention also provides a kit for determining the level of an RNA-binding compound of interest in a subject or sample, comprising (i) a large RNA target; and (ii) a fluorescent reporter molecule wherein the reporter molecule is an oxazolidinone or an aminoglycoside.  
     
    
    
     BRIEF DESCRIPTION OF DRAWINGS  
     [0074]FIG. 1 shows the structure of TAMRA-labelled oxazolidinone.  
     [0075]FIG. 2 shows the results of a ribosome binding assay using TAMRA-labelled oxazolidinone, with the circles (∘) showing % binding.  
     [0076]FIG. 3 shows the site and structure of the E-site RNA within the rRNA, and FIG. 4 shows the titration of TAMRA-labelled oxazolidinone against the E-site. FIG. 5 shows similar data for binding to rRNA.  
     [0077]FIG. 6 shows the competitive binding data for labelled (∘) and non-labelled () oxazolidinone.  
     [0078]FIG. 7 shows data from a similar competitive binding experiment using labelled oxazolidinone (∘) and linezolid ().  
     [0079]FIG. 8 shows the results of a ribosome binding assay using TAMRA-labelled paromomycin. 
    
    
     MODES FOR CARRYING OUT TILE INVENTION  
     EXAMPLE 1  
     [0080] Preparation of Oxazolidinone Reporter  
     [0081] TAMRA-labelled oxazolidinone (FIG. 1) was synthesised by reacting 4 mg oxazolidinone hydrochloride in sodium bicarbonate (6 mL 0.067M in 30% dimethyl formamide (DMF)) with 5 mg 5-carboxytetramethyl rhodamine (in 1 mL DMF) over 12 hours at room temp. The solution was diluted and purified by anion exchange chromatography and reverse phase HPLC.  
     EXAMPLE 2  
     [0082] Binding Assay with Oxazolidinone Reporter and Ribosomes  
     [0083] The interaction between oxazolidinone-TAMRA and  E. coli  ribosomes was monitored by measuring the change in fluorescence anisotropy. Each measurement was made in a 400 μL cuvette, in a Perkin Elmer LS50B fluorimeter. Increasing amounts of ribosomes (corresponding to the amounts shown in FIG. 2) were added to a solution of 50 nM oxazolidinone-TAMRA in the presence of 50 mM Tris.HCl pH 7.5, 70 mM NH 4 Cl, 30 mM KCl, 7 mM MgCl 2 , 1 mM DTT, 0.5 mM EDTA. For each titration point an anisotropy measurement was acquired using an excitation wavelength of 554 nm with the excitation slits set to 10 nm and an emission wavelength of 575 nm with the emission slits set to 10 nm. The values presented in FIG. 2 are the average of three fluorescence anisotropy measurements expressed as a percentage of the maximum anisotropy value.  
     [0084] The anisotropy shows an increase upon addition of the ribosomes. This is consistent with binding of the TAMRA-oxazolidinone to the ribosome, with a dissociation constant of 720 nM.  
     EXAMPLE 3  
     [0085] Binding Assay with Oxazolidinone Reporter and E-Site RNA  
     [0086] The interaction between oxazolidinone-TAMRA and a small  E. coli  rRNA sequence that has been identified as the binding site for the oxazolidinones (E-site RNA—FIG. 3) was monitored by measuring the change in fluorescence anisotropy. Each measurement was made in a 400 μL cuvette, in a Perkin Elmer LS50B fluorimeter, increasing amounts of E site RNA, an 82 nucleotide oligoribonucleotide (corresponding to the amounts shown in FIG. 4) were added to a solution of 50 nM oxazolidinone-TAMRA in the presence of 50 mM Tris.HCl pH7.5, 70 mM NH 4 Cl, 30 mM KCl, 7 mM MgCl 2 , 1 mM DTT, 0.5 mM EDTA. For each titration point an anisotropy measurement was acquired using an excitation wavelength of 554 nm with the excitation slits set to 10 nm and an emission wavelength of 575 nm with the emission slits set to 10 nm. The values presented were the average of three fluorescence anisotropy measurements expressed as a percentage of the maximum anisotropy value.  
     [0087] The anisotropy shows no increase upon addition of the E-site RNA. This is consistent with non-recognition of the small ‘minimal’ E-site RNA by oxazolidinone-TAMRA. Thus the trend in references 1 and 2 towards using small RNA molecules for binding studies is not suitable for studying the interaction of oxazolidinones with ribosomes—whereas oxazolidinones bind to the whole ribosome at the E-site, they do not bind to the E-site in isolation.  
     EXAMPLE 4  
     [0088] Binding Assay with Oxazolidinone Reporter and rRNA  
     [0089] The interaction between oxazolidinone-TAMRA and  E. coli  ribosomal RNA was monitored by measuring the change in fluorescence anisotropy. Each measurement was made in a 400 μL cuvette, in a Perkin Elmer LS50B fluorimeter, increasing amounts of ribosomal RNA (corresponding to the amounts shown in FIG. 5) were added to a solution of 50 nM oxazolidinone-TAMRA in the presence of 50 mM Tris.HCl pH7.5, 70 mM NH 4 Cl, 30 mM KCl, 7 mM MgCl 2 , 1 mM DT, 0.5 mM EDTA. For each titration point an anisotropy measurement was acquired using an excitation wavelength of 554 nm with the excitation slits set to 5 nm and an emission wavelength of 575 nm with the emission slits set to 10 nm. The fluorescence anisotropy values presented were the average of three measurements.  
     [0090] In contrast to the minimal ‘E-site’ RNA, the measured anisotropy shows an increase in fluorescence anisotropy upon addition of the ribosomal RNA. This is consistent with binding of the oxazolidinone-TAMRA to the ribosomal RNA, with a dissociation constant of 1600 nM. Thus a small RNA is unsuitable for studying the interaction of oxazolidinones with ribosomes, but the complete rRNA can be used.  
     EXAMPLE 5  
     [0091] Competition Assays with Oxazolidinone Reporter and Ribosomes  
     [0092] The interaction between oxazolidinone-TAMRA and  E. coli  ribosomes was monitored by comparing the change in fluorescence anisotropy in the presence and absence of 100 μM non-fluorescent oxazolidinone competitor. Each measurement was made in a 400 μl cuvette, in a Perkin Elmer LS50B fluorimeter. Increasing amounts of ribosomes (corresponding to the amounts shown in FIG. 6) were added to a solution of 20 nM oxazolidinone-TAMRA in the presence of 50 mM Tris.HCl pH 7.5, 70 mM NH 4 Cl, 30 mM KCl, 7 mM MgCl 2 , 1 mM DTT, 0.5 mM EDTA. For each titration point an anisotropy measurement was acquired using an excitation wavelength of 554 nm with the excitation slits set to 10 nm and an emission wavelength of 575 nm with the emission slits set to 10 nm. The values presented in FIG. 6 are the average of three fluorescence anisotropy measurements expressed as a percentage of the maximum anisotropy.  
     [0093] In the presence of non-fluorescent oxazolidinone competitor, the anisotropy is considerably reduced upon addition of the ribosomes. This is consistent with inhibition of the binding of TAMRA-oxazolidinone to the ribosome by the non-fluorescent oxazolidinone competitor.  
     [0094] The same experiment was performed, but using 20 nM non-fluorescent linezolid (an oxazolidinone) as the competitor. In the presence of the competitor the anisotropy is considerably reduced upon addition of the ribosomes. This is consistent with inhibition of the binding of TAMRA-oxazolidinone to the ribosome by linezolid.  
     EXAMPLE 6  
     [0095] Preparation of Paromomycin Reporter  
     [0096] TAMRA-labelled paromomycin was synthesised by reacting 55 mg paromomycin sulphate in sodium bicarbonate (6 mL 0.067M in. 30% DMF) with 5 mg 5-carboxytetramethyl rhodamine (in 1 mL DMF) over 12 hours at room temp. The solution was diluted and purified by anion exchange chromatography, and reverse phase HPLC [23].  
     EXAMPLE 7  
     [0097] Binding Assay with Paromomycin Reporter and Ribosomes  
     [0098] The interaction between paromomycin-TAMRA and  E. coli  ribosomes was monitored by measuring the change in fluorescence anisotropy. Each measurement was made in a 400 μL cuvette, in a Perkin Elmer LS50B fluorimeter. Increasing amounts of ribosomes (corresponding to the amounts shown in FIG. 8) were added to a solution of 50 nM paromomycin-TAMRA in the presence of 50 mM Tris.HCl pH 7.5, 70 mM NH 4 Cl, 30 mM KCl, 7 mM MgCl 2 , 1 nM DTT, 0.5 mM EDTA. For each titration point an anisotropy measurement was acquired using an excitation wavelength of 554 nm with the excitation slits set to 5 nm and an emission wavelength of 575 nm with the emission slits set to 10 nm. The fluorescence anisotropy values in FIG. 8 presented are the average of three measurements.  
     [0099] The measured anisotropy shows an increase in fluorescence anisotropy upon addition of the ribosomes. This is consistent with binding of the TAMRA-paromomycin to the ribosome, with a dissociation constant of 330 nM.  
     [0100] It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.  
     [0101] References  
     [0102] All references are incorporated herein by reference.  
     [0103] [1] WO99164625 (RiboTargets)  
     [0104] [2] U.S. Pat. No. 5,593,835  
     [0105] [3] Matassova et al. (1999)  RNA  5:939-946  
     [0106] [4] Brickner et al., (1996)  J. Med Chem.  39:673-79  
     [0107] [5] Tucker et al. (1998)  J. Med. Chem.  41:3727-3735  
     [0108] [6] Barbachyn et al.  Bioorganic  &amp;  Medicinal Chemistry Letters  6(9): 1003-1008  
     [0109] [7] Barbachyn et al.  Bioorganic  &amp;  Medicinal Chemistry Letters  6(9): 1009-1014  
     [0110] [8] Pae et al. (1999)  Bioorganic  &amp;  Medicinal Chemistry Letters  9:2679-2684  
     [0111] [9] Pae et al. (1999)  Bioorganic  &amp;  Medicinal Chemistry Letters  9:2685-2690  
     [0112] [10] Rheinberger et al. (1981)  PNAS USA  78:53 10-14  
     [0113] [11]  J Infect Dis  1998 Jun;177(6):1573-81  
     [0114] [12]  EMBO J  1991 Oct;10(10):3099-103  
     [0115] [13]  Biochemistry  1998 Jan 13;37(2):656-63  
     [0116] [14]  EMBO J  1998 Nov 16;17(22):6437-48  
     [0117] [15]  Biochemistry  1984 Mar 27;23(7):1462-7  
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     [0120] [18]  J Mol Biol.  1998 Mar 27;277(2):333-45  
     [0121] [19]  J Bacteriol.  1992 Dec; 174(23):7868-72.  
     [0122] [20]  J Mol Biol.  1997 Aug 29;271(4):566-87  
     [0123] [21]  Nature  1987, 327, 389-394  
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     [0126] [25]  Riedl  &amp;  Endermann, Exp. Opin. Ther. Patents  (1999), 2(5), 625-633  
     [0127] [26]  Genin, Exp. Opin. Ther. Patents  (2000), 10(1), 1405-1414  
     [0128] 
    
     
       
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             Escherichia coli  
           
            1 

uugagagaac ucgggugaag gaacuaggca aaauggugcc guaacuucgg gagaaggcac     60 

gcugauaugu aggugagguc ccucgcggau ggagcugaaa ucagucgaag auaccagcug    120 

gcugcaacug uuuauuaaaa acacagcacu gugcaaacac gaaaguggac guauacggug    180 

ugacgccugc ccggugccgg aagguuaauu gaugggguua gcgcaagcga agcucuugau    240 

cgaagccccg guaaacggcg gccguaacua uaacgguccu aagguagcga aauuccuugu    300 

cggguaaguu ccgaccugca cgaauggcgu aaugauggcc aggcugucuc cacccgagac    360 

ucagugaaau ugaacucgcu gugaagaugc aguguacccg cggcaagacg gaaagacccc    420 

gugaaccuuu acuauagcuu gacacugaac auugagccuu gauguguagg auagguggga    480 

ggcuuugaag uguggacgcc agucugcaug gagccgaccu ugaaauacca cccuuuaaug    540 

uuugauguuc uaacguugac ccguaauccg gguugcggac agugucuggu ggguaguuug    600 

acuggggcgg ucuccuccua aagaguaacg gaggagcacg aagguuggcu aauccugguc    660 

ggacaucagg agguuagugc aauggcauaa gccagcuuga cugcgagcgu gacggcgcga    720 

gcaggugcga aagcagguca uagugauccg gugguucuga auggaagggc caucgcucaa    780 

cggauaaaag guacuccggg gauaacaggc ugauaccgcc caagaguuca uaucgacggc    840 

gguguuuggc accucgaugu cggcucauca cauccugggg cugaaguagg ucccaagggu    900 

auggcuguuc gccauuuaaa gugguacgcg agcuggguuu agaacgucgu gagacaguuc    960 

ggucccuauc ugccgugggc gcuggagaac ugaggggggc ugcuccuagu acgagaggac   1020 

cggaguggac gcaucacugg uguucggguu gucaugccaa uggcacugcc cgguagcuaa   1080 

augcggaaga gauaagugcu gaaagcaucu aagcacgaaa cuugccccga gaugaguucu   1140 

cccugacccu uuaagggucc ugaaggaacg uugaagacga cgacguugau aggccgggug   1200 

uguaagcgca gcgaugcguu gagcuaaccg guacuaauga accgugaggc uuaaccuu     1258