WO2002083953A1 - Methods for identifying small molecules that bind specific rna structural motifs - Google Patents
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- WO2002083953A1 WO2002083953A1 PCT/US2002/011757 US0211757W WO02083953A1 WO 2002083953 A1 WO2002083953 A1 WO 2002083953A1 US 0211757 W US0211757 W US 0211757W WO 02083953 A1 WO02083953 A1 WO 02083953A1
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
- G01N27/44726—Arrangements for investigating the separated zones, e.g. localising zones by optical means using specific dyes, markers or binding molecules
Definitions
- the present invention relates to a method for screening and identifying test compounds that bind to a preselected target ribonucleic acid ("RNA").
- RNA ribonucleic acid
- Direct, non- competitive binding assays are advantageously used to screen libraries of compounds for those that selectively bind to a preselected target RNA. Binding of target RNA molecules to a particular test compound is detected using any physical method that measures the altered physical property of the target RNA bound to a test compound.
- the methods of the present invention provide a simple, sensitive assay for high-throughput screening of libraries of compounds to identify pharmaceutical leads.
- Protein-nucleic acid interactions are involved in many cellular functions, including transcription, RNA splicing, mRNA decay, and mRNA translation.
- Readily accessible synthetic molecules that can bind with high affinity to specific sequences of single- or double-stranded nucleic acids have the potential to interfere with these interactions in a controllable way, making them attractive tools for molecular biology and medicine.
- Successful approaches for blocking function of target nucleic acids include using duplex-forming antisense oligonucleotides (Miller, 1996, Progress in Nucl. Acid Res. & Mol. Biol.
- the antibiotic thiostreptone binds tightly to a 60-mer from ribosomal RNA (Cundliffe et al, 1990, in The Ribosome: Structure, Function & Evolution (Schlessinger et al, eds.) American Society for Microbiology, Washington, D.C. pp. 479-490). Bacterial resistance to various antibiotics often involves methylation at specific rRNA sites (Cundliffe, 1989, Ann. Rev. Microbiol. 43:207-233).
- Aminoglycosidic aminocyclitol (aminoglycoside) antibiotics and peptide antibiotics are known to inhibit group I intron splicing by binding to specific regions of the RNA (von Ahsen et al, 1991, Nature (London) 353:368-370). Some of these same aminoglycosides have also been found to inhibit hammerhead ribozyme function (Stage et al, 1995, RNA 1:95-101). In addition, certain aminoglycosides and other protein synthesis inhibitors have been found to interact with specific bases in 16S rRNA (Woodcock et al, 1991, EMBO J. 10:3099-3103).
- oligonucleotide analog of the 16S rRNA has also been shown to interact with certain aminoglycosides (Purohit et al, 1994, Nature 370:659-662).
- a molecular basis for hypersensitivity to aminoglycosides has been found to be located in a single base change in mitochondrial rRNA (Hutchin et al, 1993, Nucleic Acids Res. 21 :4174-4179).
- Aminoglycosides have also been shown to inhibit the interaction between specific structural RNA motifs and the corresponding RNA binding protein. Zapp et al.
- RNA Single stranded sections of RNA can fold into complex tertiary structures consisting of local motifs such as loops, bulges, pseudoknots, guanosine quartets and turns (Chastain & Tinoco, 1991, Progress in Nucleic Acid Res. & Mol. Biol. 41:131-177; Chow & Bogdan, 1997, Chemical Reviews 97:1489-1514; Rando & Hogan, 1998, Biologic activity of guanosine quartet forming oligonucleotides in "Applied Antisense Oligonucleotide Technology" Stein. & Krieg (eds) John Wiley and Sons, New York, pages 335-352).
- Such structures can be critical to the activity of the nucleic acid and affect functions such as regulation of mRNA transcription, stability, or translation (Weeks & Crothers, 1993, Science 261:1574-1577).
- the dependence of these functions on the native three-dimensional structural motifs of single-stranded stretches of nucleic acids makes it difficult to identify or design synthetic agents that bind to these motifs using general, simple-to-use sequence-specific recognition rules for the formation of double- and triple- helical nucleic acids used in the design of antisense and ribozyme type molecules.
- Approaches to screening generally involve competitive assays designed to identify compounds that disrupt the interaction between a target RNA and a physiological, host cell factor(s) that had been previously identified to specifically interact with that particular target RNA.
- such assays require the identification and characterization of the host cell factor(s) deemed to be required for the function of the target RNA. Both the target RNA and its preselected host cell binding partner are used in a competitive format to identify compounds that disrupt or interfere with the two components in the assay.
- the present invention relates to methods for identifying compounds that bind to preselected target elements of nucleic acids including, but not limited to, specific RNA sequences, RNA structural motifs, and/or RNA structural elements.
- the specific target RNA sequences, RNA structural motifs, and/or RNA structural elements are used as targets for screening small molecules and identifying those that directly bind these specific sequences, motifs, and/or structural elements.
- methods are described in which a preselected target RNA having a detectable label is used to screen a library of test compounds, preferably under physiologic conditions. Any complexes formed between the target RNA and a member of the library are identified using physical methods that detect the altered physical property of the target RNA bound to a test compound.
- the present invention relates to methods for using a target RNA having a detectable label to screen a library of test compounds free in solution, in labeled tubes or microtiter plate, or in a microarray.
- Compounds in the library that bind to the labeled target RNA will form a detectably labeled complex.
- the detectably labeled complex can then be identified and removed from the uncomplexed, unlabeled test compounds in the library, and from uncomplexed, labeled target RNA, by a variety of methods, including but not limited to, methods that differentiate changes in the electrophoretic, chromatographic, or thermostable properties of the complexed target RNA.
- Such methods include, but are not limited to, electrophoresis, fluorescence spectroscopy, surface plasmon resonance, mass spectrometry, scintillation, proximity assay, structure-activity relationships ("SAR") by NMR spectroscopy, size exclusion chromatography, affinity chromatography, and nanoparticle aggregation.
- SAR structure-activity relationships
- the structure of the test compound attached to the labeled RNA is then determined.
- the methods used will depend, in part, on the nature of the library screened. For example, assays or microarrays of test compounds, each having an address or identifier, may be deconvoluted, e.g., by cross-referencing the positive sample to original compound list that was applied to the individual test assays.
- test compounds identified include de novo structure determination of the test compounds using mass spectrometry or nuclear magnetic resonance ("NMR").
- NMR nuclear magnetic resonance
- the test compounds identified are useful for any purpose to which a binding reaction may be put, for example in assay methods, diagnostic procedures, cell sorting, as inhibitors of target molecule function, as probes, as sequestering agents and the like.
- small organic molecules which interact specifically with target RNA molecules may be useful as lead compounds for the development of therapeutic agents.
- the methods described herein for the identification of compounds that directly bind to a particular preselected target RNA are well suited for high-throughput screening.
- the direct binding method of the invention offers advantages over drug screening systems for competitors that inhibit the formation of naturally-occurring RNA binding proteimtarget RNA complexes; i.e., competitive assays.
- the direct binding method of the invention is rapid and can be set up to be readily performed, e.g. , by a technician, making it amenable to high throughput screening.
- the method of the invention also eliminates the bias inherent in the competitive drug screening systems, which require the use of a preselected host cell factor that may not have physiological relevance to the activity of the target RNA.
- the methods of the invention are used to identify any compound that can directly bind to specific target RNA sequences, RNA structural motifs, and/or RNA structural elements, preferably under physiologic conditions.
- the compounds so identified can inhibit the interaction of the target RNA with any one or more of the native host cell factors (whether known or unknown) required for activity of the RNA in vivo.
- a target nucleic acid refers to RNA, DNA, or a chemically modified variant thereof.
- the target nucleic acid is RNA.
- a target nucleic acid also refers to tertiary structures of the nucleic acids, such as, but not limited to loops, bulges, pseudoknots, guanosine quartets and turns.
- a target nucleic acid also refers to RNA elements such as, but not limited to, the HIV TAR element, internal ribosome entry site, "slippery site", instability elements, and adenylate uridylate-rich elements, which are described in Section 5.1. Non-limiting examples of target nucleic acids are presented in Section 5.1 and Section 6.
- a "library” refers to a plurality of test compounds with which a target nucleic acid molecule is contacted.
- a library can be a combinatorial library, e.g. , a collection of test compounds synthesized using combinatorial chemistry techniques, or a collection of unique chemicals of low molecular weight (less than 1000 daltons) that each occupy a unique three-dimensional space.
- a “label” or “detectable label” is a composition that is detectable, either directly or indirectly, by spectroscopic, photochemical, biochemical, immunochemicai, or chemical means.
- useful labels include radioactive isotopes (e.g., 32 P, 35 S, and 3 H), dyes, fluorescent dyes, electron-dense reagents, enzymes and their substrates (e.g., as commonly used in enzyme-linked immunoassays, e.g., alkaline phosphatase and horse radish peroxidase), biotin-streptavidin, digoxigenin, or haptens and proteins for which antisera or monoclonal antibodies are available.
- radioactive isotopes e.g., 32 P, 35 S, and 3 H
- dyes e.g., 32 P, 35 S, and 3 H
- dyes e.g., fluorescent dyes
- electron-dense reagents e.g., enzyme-linked
- a label or detectable moiety can include a "affinity tag" that, when coupled with the target nucleic acid and incubated with a test compound or compound library, allows for the affinity capture of the target nucleic acid along with molecules bound to the target nucleic acid.
- affinity tag bound to the target nucleic acids has, by definition, a complimentary ligand coupled to a solid support that allows for its capture.
- useful affinity tags and complimentary partners include, but are not limited to, biotin-streptavidin, complimentary nucleic acid fragments (e.g., oligo dT-oligo dA, oligo T-oligo A, oligo dG-oligo dC, oligo G-oligo C), aptamers, or haptens and proteins for which antisera or monoclonal antibodies are available.
- the label or detectable moiety is typically bound, either covalently, through a linker or chemical bound, or through ionic, van der Waals or hydrogen bonds to the molecule to be detected.
- a "dye” refers to a molecule that, when exposed to radiation, emits radiation at a level that is detectable visually or via conventional spectroscopic means.
- a "visible dye” refers to a molecule having a chromophore that absorbs radiation in the visible region of the spectrum (i.e., having a wavelength of between about 400 nm and about 700 nm) such that the transmitted radiation is in the visible region and can be detected either visually or by conventional spectroscopic means.
- an "ultraviolet dye” refers to a molecule having a chromophore that absorbs radiation in the ultraviolet region of the spectrum (i.e., having a wavelength of between about 30 nm and about 400 nm).
- an "infrared dye” refers to a molecule having a chromophore that absorbs radiation in the infrared region of the spectrum (i.e., having a wavelength between about 700 nm and about 3,000 nm).
- a “chromophore” is the network of atoms of the dye that, when exposed to radiation, emits radiation at a level that is detectable visually or via conventional spectroscopic means.
- a dye absorbs radiation in one region of the spectrum, it may emit radiation in another region of the spectrum.
- an ultraviolet dye may emit radiation in the visible region of the spectrum.
- a dye can transmit radiation or can emit radiation via fluorescence or phosphorescence.
- phrases "pharmaceutically acceptable salt(s),” as used herein includes but is not limited to salts of acidic or basic groups that may be present in test compounds identified using the methods of the present invention. Test compounds that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids.
- the acids that can be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, including but not limited to sulfuric, citric, maleic, acetic, oxalic, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pam
- Test compounds that include an amino moiety may form pharmaceutically or cosmetically acceptable salts with various amino acids, in addition to the acids mentioned above.
- Test compounds that are acidic in nature are capable of forming base salts with various pharmacologically or cosmetically acceptable cations.
- Examples of such salts include alkali metal or alkaline earth metal salts and, particularly, calcium, magnesium, sodium lithium, zinc, potassium, and iron salts.
- substantially one type of test compound is meant that the assay can be performed in such a fasliion that at some point, only one compound need be used in each reaction so that, if the result is indicative of a binding event occurring between the target RNA molecule and the test compound, the test compound can be easily identified.
- FIG. 1 Gel retardation analysis to detect peptide-RNA interactions.
- 58 peptide (0.1 ⁇ M, 0.2 ⁇ M, 0.4 ⁇ M, 0.8 ⁇ M, 1.6 ⁇ M) 50 pmole TAR RNA oligonucleotide was added in TK buffer.
- the reaction mixture was then heated at 90 °C for 2 min and allowed to cool slowly to 24 °C.
- 10 ml of 30% glycerol was added to each sample and applied to a 12% non-denaturing polyacrylamide gel.
- the gel was electrophoresed using 1200 volt-hours at 4°C in TBE Buffer.
- FIG. 2 Gentamicin interacts with an oligonucleotide corresponding to the 16S rRNA. 20 ⁇ l reactions containing increasing concentrations of gentamicin (1 ng/ml, 10 ng/ml, 100 ng/ml, 1 ⁇ g/ml, 10 ⁇ g/ml, 50 ⁇ g/ml, 500 ⁇ g/ml) were added to 50 pmole RNA oligonucleotide in TKM buffer, heated at 90 °C for 2 min and allowed to cool slowly to 24 °C.
- TAR TAR
- Tat-TAR the Tat-TAR complex
- the present invention relates to methods for identifying compounds that bind to preselected target elements of nucleic acids, in particular, RNAs, including but not limited to preselected target RNA sequencing structural motifs, or structural elements. Methods are described in which a preselected target RNA having a detectable label is used to screen a library of test compounds. Any complexes formed between the target RNA and
- RNA-test compound complex relative to the target RNA or test compound can be measured by methods such as, but not limited to, methods that detect a change in mobility due to a change in mass, change in charge, or a change in thermostability.
- the present invention relates to methods for using a target RNA having a detectable label to screen a library of test compounds free in
- test compound 30 in labeled tubes or microtiter plate, or in a microarray.
- Compounds in the library that bind to the labeled target RNA will form a detectably labeled complex.
- the detectably labeled complex can then be identified and removed from the unlabeled, uncomplexed test compounds in the library by a variety of methods capable of differentiating changes in the physical properties of the complexed target RNA.
- 35 attached to the labeled RNA is also determined.
- the methods used will depend, in part, on the nature of the library screened. For example, assays or microarrays of test compounds, each having an address or identifier, may be deconvoluted, e.g., by cross-referencing the positive sample to an original compound list that was applied to the individual test assays.
- Another method for identifying test compounds includes de novo structure determination of the test compounds using mass spectrometry or nuclear magnetic resonance ("NMR").
- the methods of the present invention provide a simple, sensitive assay for high-throughput screening of libraries of test compounds, in which the test compounds of the library that specifically bind a preselected target nucleic acid are easily distinguished from non-binding members of the library.
- the structures of the binding molecules are deciphered from the input library by methods depending on the type of library that is used.
- the test compounds so identified are useful for any purpose to which a binding reaction may be put, for example in assay methods, diagnostic procedures, cell sorting, as inhibitors of target molecule function, as probes, as sequestering agents and lead compounds for development of therapeutics, and the like. Small organic compounds that are identified to interact specifically with the target RNA molecules are particularly attractive candidates as lead compounds for the development of therapeutic agents.
- the assay of the invention reduces bias introduced by competitive binding assays which require the identification and use of a host cell factor (presumably essential for modulating RNA function) as a binding partner for the target RNA.
- the assays of the present invention are designed to detect any compound or agent that binds to the target RNA, preferably under physiologic conditions. Such agents can then be tested for biological activity, without establishing or guessing which host cell factor or factors is required for modulating the function and/or activity of the target RNA.
- Section 5.1 describes examples of protein-RNA interactions that are important in a variety of cellular functions and several target RNA elements that can be used to identify test compounds. Compounds that inhibit these interactions by binding to the RNA and successfully competing with the natural protein or host cell factor that endogenously binds to the RNA may be important, e.g. , in treating or preventing a disease or abnormal condition, such as an infection or unchecked growth.
- Section 5.2 describes detectable labels for target nucleic acids that are useful in the methods of the invention.
- Section 5.3 describes libraries of test compounds. Section 5.4 provides conditions for binding a labeled target RNA to a test compound of a library and detecting RNA binding to a test compound using the methods of the invention.
- Section 5.5 provides methods for separating complexes of target RNAs bound to a test compound from an unbound RNA.
- Section 5.6 describes methods for identifying test compounds that are bound to the target RNA.
- Section 5.7 describes a secondary, biological screen of test compounds identified by the methods of the invention to test the effect of the test compounds in vivo.
- Section 5.8 describes the use of test compounds identified by the methods of the invention for treating or preventing a disease or abnormal condition in mammals.
- Nucleic acids and in particular RNAs, are capable of folding into complex tertiary structures that include bulges, loops, triple helices and pseudoknots, which can provide binding sites for host cell factors, such as proteins and other RNAs.
- RNA-protein and RNA-RNA interactions are important in a variety cellular functions, including transcription, RNA splicing, RNA stability and translation.
- the binding of such host cell factors to RNAs may alter the stability and translational efficiency of such RNAs, and according affect subsequent translation. For example, some diseases are associated with protein overproduction or decreased protein function. In this case, the identification of compounds to modulate RNA stability and translational efficiency will be useful to treat and prevent such diseases.
- the methods of the present invention are useful for identifying test compounds that bind to target RNA elements in a high throughput screening assay of libraries of test compounds in solution.
- the methods of the present invention are useful for identifying a test compound that binds to a target RNA elements and inhibits the interaction of that RNA with one or more host cell factors in vivo.
- the molecules identified using the methods of the invention are useful for inhibiting the formation of a specific bound RNA:host cell factor complexes in vivo.
- test compounds identified by the methods of the invention are useful for increasing or decreasing the translation of messenger RNAs ("mRNAs"), e.g., protein production, by binding to one or more regulatory elements in the 5' untranslated region, the 3' untranslated region, or the coding region of the mRNA.
- mRNAs messenger RNAs
- Compounds that bind to mRNA can, inter alia, increase or decrease the rate of mRNA processing, alter its transport through the cell, prevent or enhance binding of the mRNA to ribosomes, suppressor proteins or enhancer proteins, or alter mRNA stability. Accordingly, compounds that increase or decrease mRNA translation can be used to treat or prevent disease.
- diseases associated with protein overproduction such as amyloidosis, or with the production of mutant proteins, such as Ras
- diseases associated with protein overproduction can be treated or prevented by decreasing translation of the mRNA that codes for the overproduced protein, thus inhibiting production of the protein.
- the symptoms of diseases associated with decreased protein function such as hemophelia, may be treated by increasing translation of mRNA coding for the protein whose function is decreased, e.g., factor IX in some forms of hemophilia.
- the methods of the invention can be used to identify compounds that bind to mRNAs coding for a variety of proteins with which the progression of diseases in mammals is associated.
- mRNAs include, but are not limited to, those coding for amyloid protein and amyloid precursor protein; anti-angiogenic proteins such as angiostatin, endostatin, METH-1 and METH-2; apoptosis inhibitor proteins such as survivin, clotting factors such as Factor IX, Factor VIII, and others in the clotting cascade; collagens; cyclins and cyclin inhibitors, such as cyclin dependent kinases, cyclin Dl, cyclin E, WAF1, cdk4 inhibitor, and MTS1; cystic fibrosis transmembrane conductance regulator gene (CFTR); cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL
- the invention in addition to the eukaryotic genes listed above, the invention, as described, can be used to define molecules that interrupt viral, bacterial " or fungal transcription or translation efficiencies and therefore form the basis for a novel anti- infectious disease therapeutic.
- Other target genes include, but are not limited to, those disclosed in Section 5.1 and Section 6.
- the methods of the invention can be used to identify mRNA-binding test compounds for increasing or decreasing the production of a protein, thus treating or preventing a disease associated with decreasing or increasing the production of said protein, respectively.
- the methods of the invention may be useful for identifying test compounds for treating or preventing a disease in mammals, including cats, dogs, swine, horses, goats, sheep, cattle, primates and humans.
- diseases include, but are not limited to, amyloidosis, hemophilia, Alzheimer's disease, atherosclerosis, cancer, giantism, dwarfism, hypothyroidism, hyperthyroidism, inflammation, cystic fibrosis, autoimmune disorders, diabetes, aging, obesity, neurodegenerative disorders, and Parkinson's disease.
- Other diseases include, but are not limited to, those described in Section 5.1 and diseases caused by aberrant expression of the genes disclosed in Example 6.
- the invention as described, can be used to define molecules that interrupt viral, bacterial or fungal transcription or translation efficiencies and therefore form the basis for a novel anti-infectious disease therapeutic.
- test compounds identified by the methods of the invention are useful for preventing the interaction of an RNA, such as a transfer RNA ("tRNA”), an enzymatic RNA or a ribosomal RNA (“rRNA”), with a protein or with another RNA, thus preventing, e.g., assembly of an in vivo protein-RNA or RNA-RNA complex that is essential for the viability of a cell.
- RNA transfer RNA
- rRNA ribosomal RNA
- inhibition of an interaction between rRNA and one or more ribosomal proteins may inhibit the assembly of ribosomes, rendering a cell incapable of synthesizing proteins.
- inhibition of the interaction of precursor rRNA with ribonucleases or ribonucleoprotein complexes (such as RNase P) that process the precursor rRNA prevent maturation of the rRNA and its assembly into ribosomes.
- a tRNA:tRNA synthetase complex may be inhibited by test compounds identified by the methods of the invention such that tRNA molecules do not become charged with amino acids.
- Such interactions include, but are not limited to, rRNA interactions with ribosomal proteins, tRNA interactions with tRNA synthetase, RNase P protein interactions with RNase P RNA, and telomerase protein interactions with telomerase RNA.
- test compounds identified by the methods of the invention are useful for treating or preventing a viral, bacterial, protozoan or fungal infection.
- transcriptional up-regulation of the genes of human immunodeficiency virus type 1 (“HIV-l ") requires binding of the HIV Tat protein to the HIV trans-activation response region RNA ("TAR RNA").
- HIV TAR RNA is a 59-base stem-loop structure located at the 5 '-end of all nascent HIV-1 transcripts (Jones & Peterlin, 1994, Annu. Rev. Biochem. 63:717-43). Tat protein is known to interact with uracil 23 in the bulge region of the stem of TAR RNA.
- TAR RNA is a potential binding target for test compounds, such as small peptides and peptide analogs that bind to the bulge region of TAR RNA and inhibit formation of a Tat-TAR RNA complex involved in HIV-1 upregulation (see Hwang et a/., 1999 Proc. Natl. Acad. Sci. USA 96:12997-13002). Accordingly, test compounds that bind to TAR RNA are useful as anti-HIV therapeutics (Hamy et al, 1997, Proc. Natl. Acad. Sci. USA 94:3548-3553; Hamy et al, 1998, Biochemistry 37:5086-5095; Mei et al, 1998, Biochemistry 37:14204-14212), and therefore, are useful for treating or preventing AIDS.
- test compounds such as small peptides and peptide analogs that bind to the bulge region of TAR RNA and inhibit formation of a Tat-TAR RNA complex involved in HIV-1 upregulation (see Hwang et a/
- the methods of the invention can be used to identify test compounds to treat or prevent viral, bacterial, protozoan or fungal infections in a patient.
- the methods of the invention are useful for identifying compounds that decrease translation of microbial genes by interacting with mRNA, as described above, or for identifying compounds that inhibit the interactions of microbial RNAs with proteins or other ligands that are essential for viability of the virus or microbe.
- microbial target RNAs useful in the present invention for identifying antiviral, antibacterial, anti- protozoan and anti-fungal compounds include, but are not limited to, general antiviral and
- anti-inflammatory targets such as mRNAs of INF ⁇ , INF ⁇ , RNAse L, RNAse L inhibitor protein, PKR, tumor necrosis factor, interleukins 1-15, and IMP dehydrogenase; internal ribosome entry sites; HIV-1 CT rich domain and RNase H mRNA; HCV internal ribosome entry site (required to direct translation of HCV mRNA), and the 3 '-untranslated tail of HCV genomes; rotavirus NSP3 binding site, which binds the protein NSP3 that is required
- RNA component of telomerase in fungi and cancer cells.
- target viral and bacterial mRNAs include, but are not limited to, those disclosed in Section 6.
- RNAs are functionally conserved in various species (e.g., from yeast to humans), they exhibit nucleotide sequence and structural diversity. Therefore, inhibition of, for example, yeast telomerase by an anti-fungal compound identified by the methods of the invention might not interfere with human telomerase and normal human cell proliferation.
- test compounds that interfere with one or more target RNA interactions with host cell factors that are important for cell growth or viability, or essential in the life cycle of a virus, a bacterium, a protozoa or a fungus.
- test compounds and/or congeners that demonstrate desirable biologic and pharmacologic activity can be administered to a patient in need thereof in order to treat or prevent a disease caused by viral, bacterial, protozoan, or fungal infections.
- Such diseases include, but are not limited to, HIV infection, AIDS, human T-cell leukemia, SIV infection, FIV infection, feline leukemia, hepatitis A, hepatitis B, hepatitis C, Dengue fever, malaria, rotavirus infection, severe acute gastroenteritis, diarrhea, encephalitis, hemorrhagic fever, syphilis, legionella, whooping cough, gonorrhea, sepsis, influenza, pneumonia, tinea infection, Candida infection, and meningitis.
- Non-limiting examples of RNA elements involved in the regulation of gene expression include the HIV TAR element, internal ribosome entry site, "slippery site", instability elements, and adenylate uridylate-rich elements, as discussed below.
- HIV-1 human immunodeficiency virus type 1
- TAR RNA HIV trans-activation response region RNA
- Tat protein is known to interact with uracil 23 in the bulge region of the stem of TAR RNA.
- TAR RNA is a useful binding target for test compounds, such as small peptides and peptide analogs that bind to the bulge region of TAR RNA and inhibit formation of a Tat- TAR RNA complex involved in HIV-1 up-regulation (see Hwang et al.,1999 Proc. Natl. Acad. Sci. USA 96:12997-13002). Accordingly, test compounds that bind to TAR RNA can be useful as anti-HIV therapeutics (Hamy et al, 1997, Proc. Natl. Acad. Sci. USA 94:3548-3553; Hamy et al, 1998, Biochemistry 37:5086-5095; Mei et al, 1998, Biochemistry 37:14204-14212), and therefore, are useful for treating or preventing AIDS.
- test compounds such as small peptides and peptide analogs that bind to the bulge region of TAR RNA and inhibit formation of a Tat- TAR RNA complex involved in HIV-1 up-regulation (see Hwang e
- IRES Internal ribosome entry sites
- a large segment of the 5' nontranslated region approximately 400 nucleotides in length, promotes internal entry of ribosomes independent of the non-capped 5' end of picornavirus mRNAs (mammalian plus-strand RNA viruses whose genomes serve as mRNA).
- This 400 nucleotide segment maps approximately 200 nt down-stream from the 5' end and is highly structured. IRES elements of different picornaviruses, although functionally similar in vitro and in vivo, are not identical in sequence or structure.
- the IRES elements of cardio-, entero- and aphthoviruses bind a cellular protein, p57. In the case of cardioviruses, the interaction between a specific stem-loop of the IREs is essential for translation in vitro.
- IRES elements of entero- and cardioviruses also bind the cellular protein, p52, but the significance of this interaction remains to be shown.
- the function of p57 or p52 in cellular metabolism is unknown. Since picornaviral IRES elements function in vivo in the absence of any viral gene products, is speculated that IRES-like elements may also occur in specific cellular mRNAs releasing them from cap-dependent translation (Jang et al, 1990, Enzyme 44(l-4):292-309).
- ribosomal frameshifting when ribosomes shift from one translation reading frame to another and synthesize two viral proteins from a single viral mRNA, is directed by a unique site in viral mRNAs called the "slippery site.”
- the slippery site directs ribosomal frameshifting in the -1 or +1 direction that causes the ribosome to slip by one base in the 5' direction thereby placing the ribosome in the new reading frame to produce a new protein.
- Programmed, or directed, ribosomal frameshifting is of particular value to viruses that package their plus strands, as it eliminates the need to splice their mRNAs and reduces the risk of packaging defective genomes and regulates the ratio of viral proteins synthesized.
- Examples of programmed translational frameshifting (both +1 and -1 shifts) have been identified in ScV systems (Lopinski et al, 2000, Mol. Cell. Biol. 20(4):1095-103, retroviruses (Falk et al, 1993, J. Virol.
- Drugs targeted to ribosomal frameshifting minimize the problem of virus drug resistance because this strategy targets a host cellular process rather than one introduced into the cell by the virus, which minimizes the ability of viruses to evolve drug- resistant mutants.
- Compounds that target the RNA elements involved in regulating programmed frameshifting should have several advantages, including (a) any selective pressure on the host cellular translational machinery to adapt to the drugs would have to occur at the host evolutionary time scale, which is on the order of millions of years, (b) ribosomal frameshifting is not used to express any host proteins, and (c) altering viral frameshifting efficiencies by modulating the activity of a host protein minimizing the likelihood that the virus will acquire resistance to such inhibition by mutations in its own genome.
- Instability elements may be defined as specific sequence elements that promote the recognition of unstable mRNAs by cellular turnover machinery. Instability elements have been found within mRNA protein coding regions as well as untranslated regions.
- mRNA stability may lead to disease.
- the alteration of mRNA stability has been implicated in diseases such as, but not limited to, cancer, immune disorders, heart disease, and fibrotic disorders.
- the highly oncogenic v-fos mRNA lacks the 3' UTR adenylate uridylate rich element ("ARE") that is found in the more labile and weakly oncogenic c-fos mRNA (see, e.g., Schiavi et al, 1992, Biochim Biophys Acta. 1114(2-3):95-106). Differences between the benign cervical lesions brought about by nonintegrated circular human papillomavirus type 16 and its integrated form, that lacks the 3' UTR ARE and correlates with cervical carcinomas, may be a consequence of stabilizing the E6/E7 transcripts encoding oncogenic proteins.
- ARE 3' UTR adenylate uridylate rich element
- ARE instability element results in deletion of the ARE instability element, resulting in stabilizion of the transcripts and over- expression of the proteins (see, e.g., Jeon & Lambert, 1995, Proc. Natl. Acad. Sci. USA 92(5):1654-8).
- Deletion of AREs from the 3' UTR of the IL-2 and IL-3 genes promotes increased stabilization of these mRNAs, high expression of these proteins, and leads to the formation of cancerous cells (see, e.g., Stoecklin et al, 2000, Mol. Cell. Biol. 20(11 ):3753-63).
- Mutations in trans-acting factors involved in mRNA turnover may also promote cancer.
- the lymphokine GM-CSF mRNA is specifically stabilized as a consequence of an oncogenic lesion in a trans-acting factor that controls mRNA turnover rates.
- the normally unstable IL-3 transcript is inappropriately long-lived in mast tumor cells.
- the labile GM-CSF mRNA is greatly stabilized in bladder carcinoma cells. See, e.g., Bickel et al, 1990, J. Immunol. 145(3):840-5.
- the immune system is regulated by a large number of regulatory molecules that either activate or inhibit the immune response. It has now been clearly demonstrated that stability of the transcripts encoding these proteins are highly regulated. Altered regulation of these molecules leads to mis-regulation of this process and can result in drastic medical consequences. For example, recent results using transgenic mice have shown that mis-regulation of the stability of the important modulator TNF ⁇ mRNA leads to diseases such as, but not limited to, rheumatoid arthritis and a Crohn's-like liver disease. See, e.g., Clark, 2000, Arthritis Res. 2(3): 172-4.
- Smooth muscle in the heart is modulated by the ⁇ -adrenergic receptor, which in turn responds to the sympathetic neurotransmitter norepinephrine and the adrenal hormone epinephrine.
- Chronic heart failure is characterized by impairment of smooth muscle cells, which results, in part, from the more rapid decay of the ⁇ -adrenergic receptor mRNA. See, e.g., Ellis & Frielle, 1999, Biochem. Biophys. Res. Commun. 258(3):552-8.
- a large number of diseases result from over-expression of collagen.
- cirrhosis results from damage to the liver as a consequence of cancer, viral infection, or alcohol abuse. Such damage causes mis-regulation of collagen expression, leading to the formation of large collagen deposits. Recent results indicate that the sizeable increase in collagen expression is largely attributable to stabilization of its mRNA. See, e.g., Lindquist et al, 2000, Am. J. Physiol. Gastrointest. Liver Physiol. 279(3):G471-6.
- ARE Adenylate uridylate-rich elements
- 3' UTR 3' untranslated regions
- AREs may function both
- ARE mRNAs are classified into five groups, depending on sequence (Bakheet et al, 2001, Nucl. Acids Res. 29(l):246-254).
- An ongoing database at the web site http://rc.kfshrc.edu.sa/ared contains ARE-containing mRNAs and their cluster groups, which is incorporated by reference in its entirety.
- ARE motifs are classified as follows:
- the ARE-mRNAs were clustered into five groups containing five, four, three and two pentameric repeats, while the last group contains only one pentamer within the 13-bp ARE pattern.
- Functional categories were assigned whenever possible according to NCBI-COG functional annotation (Tatusov et al, 2001, Nucleic Acids Research, 29(1): 5 22-28), in addition to the categories: inflammation, immune response, development/differentiation, using an extensive literature search.
- Group I contains many secreted proteins including GM-CSF, IL-1, IL-11, IL-12 and Gro- ⁇ that affect the growth of hematopoietic and immune cells (Witsell & Schook, 1992, Proc. Natl Acad. Sci. USA, 89:4754-4758). Although INF ⁇ is both a
- Groups II-V contain functionally diverse gene families comprising immune response, cell cycle and proliferation, inflammation and coagulation,
- ELAV embryonic lethal abnormal vision
- AUF 1 HuR and Hel-N2
- the zinc-finger protein tristetraprolin has been identified as another ARE-binding protein with destabilizing activity on TNF ⁇ , IL-3 and GM-CSF mRNAs (Stoecklin et al, 2000, Mol. Cell. Biol.
- ARE-containing genes are clearly important in biological systems, including but not limited to a number of the early response genes that regulate cell proliferation and responses to exogenous agents, the identification of compounds that bind to one or more of the ARE clusters and potentially modulate the stability of the target RNA
- 25 can potentially be of value as a therapeutic.
- Target nucleic acids including but not limited to RNA and DNA, useful in the methods of the present invention have a label that is detectable via conventional 0 spectroscopic means or radiographic means.
- target nucleic acids are labeled with a covalently attached dye molecule.
- Useful dye-molecule labels include, but are not limited to, fluorescent dyes, phosphorescent dyes, ultraviolet dyes, infrared dyes, and visible dyes.
- the dye is a visible dye.
- Useful labels in the present invention can include, but are not limited to, 35 spectroscopic labels such as fluorescent dyes (e.g., fluorescein and derivatives such as fluorescein isothiocyanate (FITC) and Oregon GreenTM, rhodamine and derivatives (e.g., Texas red, tetramethylrhodimine isothiocynate (TRITC), bora-3a,4a-diaza-s-indacene (BODIPY®) and derivatives, etc.), digoxigenin, biotin, phycoerythrin, AMCA, CyDyeTM, and the like), radiolabels (e.g., 3 H, 125 1, 35 S, 14 C, 3 P, 33 P, etc.), enzymes (e.g., horse radish peroxidase, alkaline phosphatase etc.), spectroscopic colorimetric labels such as colloidal gold or colored glass or plastic (e.g.
- fluorescent dyes
- affinity tags and complimentary partners include, but are not limited to, biotin-streptavidin, complimentary nucleic acid fragments (e.g., oligo dT-oligo dA, oligo T-oligo A, oligo dG-oligo dC, oligo G-oligo C), aptamer-streptavidin, or haptens and proteins for which antisera or monoclonal antibodies are available.
- the label may be coupled directly or indirectly to a component of the detection assay (e.g., the detection reagent) according to methods well known in the art.
- a component of the detection assay e.g., the detection reagent
- a wide variety of labels may be used, with the choice of label depending on sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.
- nucleic acids that are labeled at one or more specific locations are chemically synthesized using phosphoramidite or other solution or solid-phase methods.
- phosphoramidite or other solution or solid-phase methods.
- Detailed descriptions of the chemistry used to form polynucleotides by the phosphoramidite method are well known (see, e.g., Caruthers et ⁇ i., U.S. Pat. Nos. 4,458,066 and 4,415,732; Caruthers et ⁇ l, 1982, Genetic Engineering 4:1-17; Users Manual Model 392 and 394 Polynucleotide Synthesizers, 1990, pages 6-1 through 6-22, Applied Biosystems, Part No.
- the phosphoramidite method of polynucleotide synthesis is the preferred method because of its efficient and rapid coupling and the stability of the starting materials.
- the synthesis is performed with the growing polynucleotide chain attached to a solid support, such that excess reagents, which are generally in the liquid phase, can be easily removed by washing, decanting, and/or filtration, thereby eliminating the need for purification steps between synthesis cycles.
- a solid support to which is attached a protected nucleoside monomer at its 3' terminus is treated with acid, e.g., trichloroacetic acid, to remove the 5 '-hydroxyl protecting group, freeing the hydroxyl group for a subsequent coupling reaction.
- acid e.g., trichloroacetic acid
- an activated intermediate is formed by contacting the support-bound nucleoside with a protected nucleoside phosphoramidite monomer and a weak acid, e.g., tetrazole.
- the weak acid protonates the nitrogen atom of the phosphoramidite forming a reactive intermediate.
- Nucleoside addition is generally complete within 30 seconds.
- a capping step is performed, which terminates any polynucleotide chains that did not undergo nucleoside addition.
- Capping is preferably performed using acetic anhydride and 1 -methylimidazole.
- the phosphite group of the internucleotide linkage is then converted to the more stable phosphotriester by oxidation using iodine as the preferred oxidizing agent and water as the oxygen donor.
- the hydroxyl protecting group of the newly added nucleoside is removed with a protic acid, e.g., trichloroacetic acid or dichloroacetic acid, and the cycle is repeated one or more times until chain elongation is complete.
- a protic acid e.g., trichloroacetic acid or dichloroacetic acid
- the polynucleotide chain is cleaved from the support using a base, e.g., ammonium hydroxide or t-butyl amine.
- a base e.g., ammonium hydroxide or t-butyl amine.
- the cleavage reaction also removes any phosphate protecting groups, e.g., cyanoethyl.
- the protecting groups on the exocyclic amines of the bases and any protecting groups on the dyes are removed by treating the polynucleotide solution in base at an elevated temperature, e.g., at about 55°C.
- the various protecting groups are removed using ammonium hydroxide or t-butyl amine.
- any of the nucleoside phosphoramidite monomers can be labeled using standard phosphoramidite chemistry methods (Hwang et al, 1999, Proc. Natl. Acad. Sci. USA 96(23):12997-13002; Ojwang et al, 1997, Biochemistry. 36:6033-6045 and references cited therein).
- Dye molecules useful for covalently coupling to phosphoramidites preferably comprise a primary hydroxyl group that is not part of the dye's chromophore.
- Illustrative dye molecules include, but are not limited to, disperse dye CAS 4439-31-0, disperse dye CAS 6054-58-6, disperse dye CAS 4392-69-2 (Sigma-Aldrich, St.
- dye-labeled target RNA molecules are synthesized enzymatically using in vitro transcription (Hwang et al, 1999, Proc. Natl. Acad. Sci. USA 96(23):12997-13002 and references cited therein).
- a template DNA is denatured by heating to about 90°C and an oligonucleotide primer is annealed to the template DNA, for example by slow-cooling the mixture of the denatured template and the primer from about 90°C to room temperature.
- a mixture of ribonucleoside-5'-triphosphates capable of supporting template-directed enzymatic extension of the primed template e.g., a mixture including GTP, ATP, CTP, and UTP
- a polymerase enzyme is added to the mixture under conditions where the polymerase enzyme is active, which are well-known to those skilled in the art.
- a labeled polynucleotide is formed by the incorporation of the labeled ribonucleotides during polymerase-mediated strand synthesis.
- nucleic acid molecules are end- labeled after their synthesis.
- Methods for labeling the 5 '-end of an oligonucleotide include but are by no means limited to: (i) periodate oxidation of a 5 '-to-5' -coupled ribonucleotide, followed by reaction with an amine-reactive label (Heller & Morisson, 1985, in Rapid Detection and Identification of Infectious Agents, D.T. Kingsbury and S. Falkow, eds., pp.
- a detectable label should not be incorporated into a target nucleic acid at the specific binding site at which test compounds are likely to bind, since the presence of a covalently attached label might interfere sterically or chemically with the binding of the test compounds at this site. Accordingly, if the region of the target nucleic acid that binds to a host cell factor is known, a detectable label is preferably incorporated into the nucleic acid molecule at one or more positions that are spatially or sequentially remote from the binding region.
- the labeled target nucleic acid can be purified using standard techniques known to those skilled in the art (see Hwang et al, 1999, Proc. Natl. Acad. Sci. USA 96(23):12997-13002 and references cited therein).
- purification techniques include, but are not limited to, reverse-phase high-performance liquid chromatography ("reverse-phase HPLC”), fast performance liquid chromatography (“FPLC”), and gel purification.
- the target RNA is refolded into its native conformation, preferably by heating to approximately 85-95°C and slowly cooling to room temperature in a buffer, e.g., a buffer comprising about 50 mM Tris-HCI, pH 8 and 100 mM NaCl.
- a buffer e.g., a buffer comprising about 50 mM Tris-HCI, pH 8 and 100 mM NaCl.
- the target nucleic acid can also be radiolabeled.
- a radiolabel such as, but not limited to, an isotope of phosphorus, sulfur, or hydrogen, may be incorporated into a nucleotide, which is added either after or during the synthesis of the target nucleic acid.
- Methods for the synthesis and purification of radiolabeled nucleic acids are well known to one of skill in the art. See, e.g., Sambrook et al, 1989, inMolecular Cloning: A Laboratory Manual, pp 10.2-10.70, Cold Spring Harbor Laboratory Press, and the references cited therein, which are hereby incorporated by reference in their entireties.
- the target nucleic acid can be attached to an inorganic nanoparticle.
- a nanoparticle is a cluster of ions with controlled size from 0.1 to 1000 mu comprised of metals, metal oxides, or semiconductors including, but not limited to Ag 2 S, ZnS, CdS, CdTe, Au, or TiO 2 . Nanoparticles have unique optical, electronic and catalytic properties relative to bulk materials which can be adjusted according to the size of the particle. Methods for the attachment of nucleic acids are well know to one of skill in the art (see, e.g., Niemeyer, 2001, Angew. Chem. Int. Ed. 40: 4129-4158, International Patent Publication WO/0218643, and the references cited therein, the disclosures of which are hereby incorporated by reference in their entireties).
- test compounds are nucleic acid or peptide molecules.
- peptide molecules can exist in a phage display library.
- types of test compounds include, but are not limited to, peptide analogs including peptides comprising non-naturally occurring amino acids, e.g., D-amino acids, phosphorous analogs of amino acids, such as ⁇ -amino phosphoric acids and ⁇ -amino phosphoric acids, or amino acids having non-peptide linkages, nucleic acid analogs such as phosphorothioates and PNAs, hormones, antigens, synthetic or naturally occurring drugs, opiates, dopamine, serotonin, catecholamines, thrombin, acetylcholine, prostaglandins, organic molecules, pheromones, adenosine, sucrose, glucose, lactose and gal
- the combinatorial libraries are small organic molecule libraries, such as, but not limited to, benzodiazepines, isoprenoids, thiazolidinones, metathiazanones, pyrrolidines, morpholino compounds, and diazepindiones.
- the combinatorial libraries comprise peptoids; random bio-oligomers; diversomers such as hydantoins, benzodiazepines and dipeptides; vinylogous polypeptides; nonpeptidal peptidomimetics; oligocarbamates; peptidyl phosphonates; peptide nucleic acid libraries; antibody libraries; or carbohydrate libraries.
- Combinatorial libraries are themselves commercially available (see, e.g., Advanced ChemTech Europe Ltd., Cambridgeshire, UK; ASINEX, Moscow Russia; BioFocus pic, Sittingbourne, UK; Bionet Research (A division of Key Organics Limited ), Camelford, UK; ChemBridge Corporation, San Diego, California; ChemDiv Inc, San Diego, California.; ChemRx Advanced Technologies, South San Francisco, California; ComGenex Inc., Budapest, Hungary; Evotec OAI Ltd, Abingdon, UK; IF LAB Ltd., Kiev, Ukraine; Maybridge pic, Cornwall, UK; PharmaCore, Inc., North Carolina; SIDDCO Inc, Arlington, Arizona; TimTec Inc, Newark, Delaware; Tripos Receptor Research Ltd, Bude, UK; Toslab,
- the combinatorial compound library for the methods of the present invention may be synthesized.
- synthetic methods directed toward the creation of large collections of small organic compounds, or libraries, which could be screened for pharmacological, biological or other activity (Dolle, 2001, J. Comb. Chem. 3:477-517; Hall et al, 2001, J. Comb. Chem. 3:125-150; Dolle, 2000, J. Comb. Chem. 2:383-433; Dolle, 1999, J. Comb. Chem. 1:235-282).
- the synthetic methods applied to create vast combinatorial libraries are performed in solution or in the solid phase, i.e., on a solid support. Solid-phase synthesis makes it easier to conduct multi-step reactions and to drive reactions to completion with high yields because excess reagents can
- Combinatorial compound libraries of the present invention may be any combination of combinatorial compound libraries of the present invention.
- the combinatorial compound library can be synthesized in solution.
- the template is designed to pe ⁇ nit reaction products to be easily purified from unreacted reactants using liquid/liquid or solid/liquid extractions.
- the compounds produced by combinatorial synthesis using the template will preferably be small organic molecules. Some compounds in the library may mimic the effects of non-peptides or peptides.
- liquid phase synthesis does not require the use of specialized protocols for monitoring the individual steps of a multistep solid phase synthesis (Egner et al, 1995, J.Org. Chem. 60:2652; Anderson et al, 1995, J. Org. Chem. 60:2650; Fitch et al, 1994, J. Org. Chem. 59:7955; Look et al, 1994, J. Org. Chem. 49:7588; Metzger et al, 1993, Angew. Chem., Int. Ed. Engl. 32:894; Youngquist et al, 1994, Rapid Commun. Mass Spect.
- Combinatorial compound libraries useful for the methods of the present invention can be synthesized on solid supports.
- a split synthesis method a protocol of separating and mixing solid supports during the synthesis, is used to synthesize a library of compounds on solid supports (see Lam et al, 1997, Chem. Rev. 97:41-448; Ohlmeyer et al, 1993, Proc. Natl. Acad. Sci. USA 90:10922-10926 and references cited therein).
- Each solid support in the final library has substantially one type of test compound attached to its surface.
- solid support is not limited to a specific type of solid support. Rather a large number of supports are available and are known to one skilled in the art. Solid supports include silica gels, resins, derivatized plastic films, glass beads, cotton, plastic beads, polystyrene beads, alumina gels, and polysaccharides. A suitable solid support may be selected on the basis of desired end use and suitability for various synthetic protocols.
- a solid support can be a resin such as p- methylbenzhydrylamine (pMBHA) resin (Peptides International, Louisville, KY), polystyrenes (e.g., PAM-resin obtained from Bachem Inc., Peninsula Laboratories, etc.), including chloromethylpolystyrene, hydroxymethylpolystyrene and aminomethylpolystyrene, poly (dimethylacrylamide)-grafted styrene co-divinyl-benzene (e.g., POLYHIPE resin, obtained from Aminotech, Canada), polyamide resin (obtained from Peninsula Laboratories), polystyrene resin grafted with polyethylene glycol (e.g., TENTAGEL or ARGOGEL, Bayer, Tubingen, Germany) polydimethylacrylamide resin (obtained from Milligen/Biosearch, California), or Sepharose (Pharmacia, Sweden).
- pMBHA p- methylbenzhydrylamine
- the solid phase support is suitable for in vivo use, i.e., it can serve as a carrier or support for administration of the test compound to a patient (e.g., TENTAGEL, Bayer, Tubingen, Germany).
- the solid support is palatable and/or orally ingestable.
- compounds can be attached to solid supports via linkers.
- Linkers can be integral and part of the solid support, or they may be nonintegral that are either synthesized on the solid support or attached thereto after synthesis.
- Linkers are useful not only for providing points of test compound attachment to the solid support, but also for allowing different groups of molecules to be cleaved from the solid support under different conditions, depending on the nature of the linker.
- linkers can be, inter alia, electrophilically cleaved, nucleophilically cleaved, photocleavable, enzymatically cleaved, cleaved by metals, cleaved under reductive conditions or cleaved under oxidative conditions.
- the combinatorial compound libraries can be assembled in situ using dynamic combinatorial chemistry as described in European Patent Application 1,118,359 Al to Lehn; Hue & Nguyen, 2001, Comb. Chem. High Throughput. Screen. 4:53-74; Lehn and Eliseev, 2001, Science 291:2331-2332; Cousins etal. 2000, Curr. Opin. Chem. Biol. 4: 270-279; and Karan & Miller, 2000, Drug. Disc. Today 5:67-75 which are incorporated by reference in their entirety.
- Dynamic combinatorial chemistry uses non-covalent interaction with a target biomolecule, including but not limited to a protein, RNA, or DNA, to favor assembly of the most tightly binding molecule that is a combination of constituent subunits present as a mixture in the presence of the biomolecule.
- a target biomolecule including but not limited to a protein, RNA, or DNA
- molecules, preferably one molecule, that bind most tightly to a templating biomolecule will be present in greater amount than all other possible combinations.
- the reversible chemical reactions include, but are not limited to, imine, acyl-hydrazone, amide, acetal, or ester formation between carbonyl-containing compounds and amines, hydrazines, or alcohols; thiol exchange between disulfides; alcohol exchange in borate esters; Diels- Alder reactions; thermal- or photoinduced sigmatropic or electrocyclic rearrangements; or Michael reactions.
- the constituent components of the dynamic combinatorial compound library are allowed to combine and reach equilibrium in the absence of the target RNA and then incubated in the presence of the target RNA, preferably at physiological conditions, until a second equilibrium is reached.
- the second, perturbed, equilibrium (the so-called “templated mixture”) can, but need not necessarily, be fixed by a further chemical transformation, including but not limited to reduction, oxidation, hydrolysis, acidification, or basification, to prevent restoration of the original equilibrium when the dynamical combinatorial compound library is separated from the target RNA.
- the predominant product or products of the templated dynamic combinatorial library can separated from the minor products and directly identified.
- the identity of the predominant product or products can be identified by a deconvolution strategy involving preparation of derivative dynamic combinatorial libraries, as described in European Patent Application 1,118,359 Al, which is incorporated by reference in their entirety, whereby each component of the mixture is, preferably one-by-one but possibly group-wise, left out of the mixture and the ability of the derivative library mixture at chemical equilibrium to bind the target RNA is measured.
- the components whose removal most greatly reduces the ability of the derivative dynamic combinatorial library to bind the target RNA are likely the components of the predominant product or products in the original dynamic combinatorial library.
- a target nucleic acid such as but not limited to RNA or DNA
- a test compound library is synthesized or purchased or both
- the labeled target nucleic acid is used to screen the library to identify test compounds that bind to the nucleic acid.
- Screening comprises contacting a labeled target nucleic acid with an individual, or small group, of the components of the compound library.
- the contacting occurs in an aqueous solution, and most preferably, under physiologic conditions.
- the aqueous solution preferably stabilizes the labeled target nucleic acid and prevents denaturation or degradation of the nucleic acid without interfering with binding of the test compounds.
- the aqueous solution can be similar to the solution in which a complex between the target RNA and its corresponding host cell factor (if known) is formed in vitr a o.
- TK buffer which is commonly used to form Tat protein-TAR RNA complexes in vitro, can be used in the methods of the invention as an aqueous solution to screen a library of test compounds for TAR RNA binding compounds.
- the methods of the present invention for screening a library of test compounds preferably comprise contacting a test compound with a target nucleic acid in the presence of an aqueous solution, the aqueous solution comprising a buffer and a combination of salts, preferably approximating or mimicking physiologic conditions.
- the aqueous solution optionally further comprises non-specific nucleic acids, such as, but not limited to, DNA; yeast tRNA; salmon sperm DNA; homoribopolymers such as, but not limited to, poly IC, polyA, polyU, and polyC; and non-specific RNA.
- the non-specific RNA may be an unlabeled target nucleic acid having a mutation at the binding site, which renders the unlabeled nucleic acid incapable of interacting with a test compound at that site.
- unlabeled TAR RNA having a mutation in the uracil 23/cytosine 24 bulge region may also be present in the aqueous solution.
- the addition of unlabeled RNA that is essentially identical to the dye-labeled target RNA except for a mutation at the binding site might minimize interactions of other regions of the dye-labeled target RNA with test compounds or with the solid support and prevent false positive results.
- the solution further comprises a buffer, a combination of salts, and optionally, a detergent or a surfactant.
- the pH of the solution typically ranges from about 5 to about 8, preferably from about 6 to about 8, most preferably from about 6.5 to about 8.
- a variety of buffers may be used to achieve the desired pH. Suitable buffers include, but are not limited to, Tris, Mes, Bis-Tris, Ada, Aces, Pipes, Mopso, Bis-Tris propane, Bes, Mops, Tes, Hepes, Dipso, Mobs, Tapso, Trizma, Heppso, Popso, TEA, Epps, Tricine, Gly- Gly, Bicine, and sodium-potassium phosphate.
- the buffering agent comprises from about 10 mM to about 100 mM, preferably from about 25 mM to about 75 mM, most preferably from about 40 mM to about 60 mM buffering agent.
- the pH of the aqeuous solution can be optimized for different screening reactions, depending on the target RNA used and the types of test compounds in the library, and therefore, the type and amount of the buffer used in the solution can vary from screen to screen.
- the aqueous solution has a pH of about 7.4, which can be achieved using about 50 mM Tris buffer.
- the aqueous solution further comprises a combination of salts, from about 0 mM to about 100 mM KCl, from about 0 mM to about 1 M NaCl, and from about 0 mM to about 200 mM MgCl 2 .
- the combination of salts is about 100 mM KCl, 500 mM NaCl, and 10 mM MgCl 2 .
- Applicant has found that a combination of KCl, NaCl, and MgCl 2 stabilizes the target RNA such that most of the RNA is not denatured or digested over the course of the screening reaction.
- the optional concentration of each salt used in the aqueous solution is dependent on the particular target RNA used and can be determined using routine experimentation.
- the solution optionally comprises from about 0.01% to about 0.5% (w/v) of a detergent or a surfactant.
- a small amount of detergent or surfactant in the solution might reduce non-specific binding of the target RNA to the solid support and control aggregation and increase stability of target RNA molecules.
- Typical detergents useful in the methods of the present invention include, but are not limited to, anionic detergents, such as salts of deoxycholic acid, 1-heptanesulfonic acid, N- laurylsarcosine, lauryl sulfate, 1 -octane sulfonic acid and taurocholic acid; cationic detergents such as benzalkonium chloride, cetylpyridinium, methylbenzethonium chloride, and decamethonium bromide; zwitterionic detergents such as CHAPS, CHAPSO, alkyl betaines, alkyl amidoalkyl betaines, N-dodecyl-N,N-dimethyl-3-ammonio-l- propanesulfonate, and phosphatidylcholine; and non-ionic detergents such as n-decyl a-D- glucopyranoside, n-decyl ⁇ -D-maltopyranoside,
- the detergent if present, is a nonionic detergent.
- Typical surfactants useful in the methods of the present invention include, but are not limited to, ammonium lauryl sulfate, polyethylene glycols, butyl glucoside, decyl glucoside, Polysorbate 80, lauric acid, myristic acid, palmitic acid, potassium palmitate, undecanoic acid, lauryl betaine, and lauryl alcohol. More preferably, the detergent, if present, is Triton X-100 and present in an amount of about 0.1% (w/v).
- Non-specific binding of a labeled target nucleic acid to test compounds can be further minimized by treating the binding reaction with one or more blocking agents.
- the binding reactions are treated with a blocking agent, e.g., bovine serum albumin ("BSA"), before contacting with to the labeled target nucleic acid.
- BSA bovine serum albumin
- the binding reactions are treated sequentially with at least two different blocking agents. This blocking step is preferably performed at room temperature for from about 0.5 to about 3 hours.
- the reaction mixture is further treated with unlabeled RNA having a mutation at the binding site.
- This blocking step is preferably performed at about 4°C for from about 12 hours to about 36 hours before addition of the dye-labeled target RNA.
- the solution used in the one or more blocking steps is substantially similar to the aqueous solution used to screen the library with the dye-labeled target RNA, e.g. , in pH and salt concentration.
- the mixture of labeled target nucleic acid and the test compound is preferably maintained at 4°C for from about 1 day to about 5 days, preferably from about 2 days to about 3 days with constant agitation.
- bound from free compounds are determined using an electrophoretic technique (see Section 5.5.1), or any of the methods disclosed in Section 5.5 infra.
- the complexed target nucleic acid does not need to be separated from the free target nucleic acid if a technique (i.e., spectrometry) that diferentiates between bound and unbound target nucleic acids is used.
- the methods for identifying small molecules bound to labeled nucleic acid will vary with the type of label on the target nucleic acid.
- the target RNA complexes are preferably identified using a chromatographic technique that separates bound from free target by an electrophoretic or size differential technique using individual reactions.
- the reactions corresponding to changes in the migration of the complexed RNA can be cross-referenced to the small molecule compound(s) added to said reaction.
- complexed target RNA can be screened en masse and then separated from free target RNA using an electrophoretic or size differential technique, the resultant complexed target is then analyzed using a mass spectrometric technique.
- test compounds bound to the target nucleic acid may not require separation from the unbound target nucleic acid if a technique such as, but not limited to, spectrometry is used.
- Any method that detects an altered physical property of a target nucleic acid complexed to a test compound from the unbound target nucleic acid may be used for separation of the complexed and non-complexed target nucleic acids.
- Methods that can be utilized for the physical separation of complexed target RNA from unbound target RNA include, but are not limited to, electrophoresis, fluorescence spectroscopy, surface plasmon resonance, mass spectrometry, scintillation, proximity assay, structure-activity relationships ("SAR”) by NMR spectroscopy, size exclusion chromatography, affinity chromatography, and nanoparticle aggregation.
- Methods for separation of the complex of a target RNA bound to a test compound from the unbound RNA comprises any method of electrophoretic separation, including but not limited to, denaturing and non-denaturing polyacrylamide gel electrophoresis, urea gel electrophoresis, gel filtration, pulsed field gel electrophoresis, two dimensional gel electrophoresis, continuous flow electrophoresis, zone electrophoresis, agarose gel electrophoresis, and capillary electrophoresis.
- an automated electrophoretic system comprising a capillary cartridge having a plurality of capillary tubes is used for high-throughput screening of test compounds bound to target RNA.
- Such an apparatus for performing automated capillary gel electrophoresis is disclosed in U.S. Patent Nos. 5,885,430; 5,916,428; 6,027,627; and 6,063,251, the disclosures of which are incorporated by reference in their entireties.
- U.S. Patent No. 5,885,430 which is incorporated by reference in its entirety, allows one to simultaneously introduce samples into a plurality of capillary tubes directly from microtiter trays having a standard size.
- U.S. Patent No. 5,885,430 discloses a disposable capillary cartridge which can be cleaned between electrophoresis runs, the cartridge having a plurality of capillary tubes. A first end of each capillary tube is retained in a mounting plate, the first ends collectively forming an array in the mounting plate. The spacing between the first ends corresponds to the spacing between the centers of the wells of a microtiter tray having a standard size.
- the cartridge is provided with a second mounting plate in which the second ends of the capillary tubes are retained.
- the second ends of the capillary tubes are arranged in an array which corresponds to the wells in the microtiter tray, which allows for each capillary tube to be isolated from its neighbors and therefore free from cross-contamination, as each end is dipped into an individual well.
- Plate holes may be provided in each mounting plate and the capillary tubes inserted through these plate holes.
- the plate holes are sealed airtight so that the side of the mounting plate having the exposed capillary ends can be pressurized.
- Application of a positive pressure in the vicinity of the capillary openings in this mounting plate allows for the introduction of air and fluids during electrophoretic operations and also can be used to force out gel and other materials from the capillary tubes during reconditioning.
- the capillary tubes may be protected from damage using a needle comprising a cannula and/or plastic tubes, and the like when they are placed in these plate holes. When metallic cannula or the like are used, they can serve as electrical contacts for current flow during electrophoresis.
- the second mounting plate In the presence of a second mounting plate, the second mounting plate is provided with plate holes through which the second ends of the capillary tubes project.
- the second mounting plate serves as a pressure containment member of a pressure cell and the second ends of the capillary tubes communicate with an internal cavity of the pressure cell.
- the pressure cell is also formed with an inlet and an outlet. Gels, buffer solutions, cleaning agents, and the like may be introduced into the internal cavity through the inlet, and each of these can simultaneously enter the second ends of the capillaries.
- the automated electrophoretic system can comprise a chip system consisting of complex designs of interconnected channels that perform and analyze enzyme reactions using part of a channel design as a tiny, continuously operating electrophoresis material, where reactions with one sample are going on in one area of the chip while electrophoretic separation of the products of another sample is taking place in a different part of the chip.
- a chip system consisting of complex designs of interconnected channels that perform and analyze enzyme reactions using part of a channel design as a tiny, continuously operating electrophoresis material, where reactions with one sample are going on in one area of the chip while electrophoretic separation of the products of another sample is taking place in a different part of the chip.
- the system disclosed in U.S. Patent No. 5,699,157 which is hereby incorporated by reference in its entirety, provides for a microfluidic system for high-speed electrophoretic analysis of subject materials for applications in the fields of chemistry, biochemistry, biotechnology, molecular biology and numerous other areas.
- the system has a channel in a substrate, a light source and a photoreceptor.
- the channel holds subject materials in solution in an electric field so that the materials move through the channel and separate into bands according to species.
- the light source excites fluorescent light in the species bands and the photoreceptor is arranged to receive the fluorescent light from the bands.
- the system further has a means for masking the channel so that the photoreceptor can receive the fluorescent light only at periodically spaced regions along the channel.
- the system also has an unit connected to analyze the modulation frequencies of light intensity received by the photoreceptor so that velocities of the bands along the channel are determined, which allows the materials to be analyzed.
- the system disclosed in U.S. Patent No. 5,699,157 also provides for a method of performing high-speed electrophoretic analysis of subject materials, which comprises the steps of holding the subject materials in solution in a channel of a microfluidic system; subjecting the materials to an electric field so that the subject materials move through the channel and separate into species bands; directing light toward the channel; receiving light from periodically spaced regions along the channel simultaneously; and analyzing the frequencies of light intensity of the received light so that velocities of the bands along the channel can be determined for analysis of said materials.
- the determination of the velocity of a species band determines the electrophoretic mobility of the species and its identification.
- U.S. Patent No. 5,842,787 which is hereby incorporated by reference in its entirety, is generally directed to devices and systems employ channels having, at least in part, depths that are varied over those which have been previously described (such as the device disclosed in U.S. Patent No. 5,699,157), wherein said channel depths provide numerous beneficial and unexpected results such as but not limited to, a reduction in sample perturbation, reduced non-specific sample mixture by diffusion, and increased resolution.
- the electrophoretic method of separation comprises polyacrylamide gel electrophoresis.
- the polyacrylamide gel electrophoresis is non-denaturing, so as to differentiate the mobilities of the target RNA bound to a test compound from free target RNA. If the polyacrylamide gel electrophoresis is denaturing, then the target RNA:test compound complex must be cross-linked prior to electrophoresis to prevent the disassociation of the target RNA from the test compound during electrophoresis.
- Such techniques are well known to one of skill in the art.
- the binding of test compounds to target nucleic acid can be detected, preferably in an automated fashion, by gel electrophoretic analysis of interference footprinting.
- RNA can be degraded at specific base sites by enzymatic methods such as ribonucleases A, U 2 , CL 3 , T l5 Phy M, and B. cereus or chemical methods such as diethylpyrocarbonate, sodium hydroxide, hydrazine, piperidine formate, dimethyl sulfate,
- cleavage sites are determined by the accessibility of particular bases to the reagent employed to initiate cleavage and, as such, is therefore is determined by the three-dimensional structure of the RNA.
- the interaction of small molecules with a target nucleic acid can change the accessibility of bases to these cleavage reagents both by causing conformational changes in the target nucleic acid or by covering a base at the binding interface.
- a test compound binds to the nucleic acid and changes the accessibility of bases to cleavage reagents, the observed cleavage pattern will change.
- This method can be used to identify and characterize the binding of small molecules to RNA as described, for example, by Prudent et al, 1995, J. Am. Chem. Soc. 117:10145-10146 and Mei et al, 1998, Biochem. 37:14204-14212.
- the detectably labeled target nucleic acid is incubated with an individual test compound and then subjected to treatment with a cleavage reagent, either enzymatic or chemical.
- a cleavage reagent either enzymatic or chemical.
- the reaction mixture can be preferably be examined directly, or treated further to isolate and concentrate the nucleic acid.
- the fragments produced are separated by electrophoresis and the pattern of cleavage can be compared to a cleavage reaction performed in the absence of test compound.
- a change in the cleavage pattern directly indicates that the test compound binds to the target nucleic acid.
- Multiple test compounds can be examined both in parallel and serially.
- electrophoretic separation examples include, but are not limited to urea gel electrophoresis, gel filtration, pulsed field gel electrophoresis, two dimensional gel electrophoresis, continuous flow electrophoresis, zone electrophoresis, and agarose gel electrophoresis.
- fluorescence polarization spectroscopy an optical detection method that can differentiate the proportion of a fluorescent molecule that is either bound or unbound in solution (e.g., the labeled target nucleic acid of the present invention), can be used to read reaction results without electrophoretic separation of the samples.
- Fluorescence polarization spectroscopy can be used to read the reaction results in the chip system disclosed in U.S. Patent Nos.
- a compound that has an affinity for the target nucleic acid of interest can be labeled with a fluorophore to screen for test compounds that bind to the target nucleic acid.
- a fluorophore for example, a pyrene-containing aminoglycoside analog was used to accurately monitor antagonist binding to a prokaryotic 16S rRNA A site (which comprises the natural target for aminoglycoside antibiotics) in a screen using a fluorescence quenching technique in a 96-well plate format (Hamasaki & Rando, 1998, Anal. Biochem. 261(2): 183-90).
- fluorescence resonance energy transfer can be used to screen for test compounds that bind to the target nucleic acid.
- FRET fluorescence resonance energy transfer
- the fluorophore on the target nucleic acid and the fluorophore on the test compounds will have overlapping excitation and emission spectra such that one fluorophore (the donor) transfers its emission energy to excite the other fluorophore (the acceptor).
- the acceptor preferably emits light of a different wavelength upon relaxing to the ground state, or relaxes non-radiatively to quench fluorescence.
- FRET Fluorescence Activated FRET
- U.S, Patent 6,337,183 to Arenas et al which is incorporated by reference in its entirety, describes a screen for compounds that bind RNA that uses FRET to measure the effect of test compounds on the stability of a target RNA molecule where the target RNA is labeled with both fluorescent acceptor and donor molecules and the distance between the two fluorophores as determined by FRET provides a measure of the folded structure of the RNA.
- Matsumoto et al. 2000, Bioorg. Med. Chem. Lett.
- both the target nucleic acid and a compound that has an affinity for the target nucleic acid of interest are labeled with fluorophores with overlapping emission and excitation spectra (donor and acceptor), including but not limited to fluorescein and derivatives, rhodamine and derivatives, cyanine dyes and derivatives, bora-3a,4a-diaza-s-indacene (BODIPY®) and derivatives, pyrene, nanoparticles, or non-fluorescent quenching molecules.
- fluorophores with overlapping emission and excitation spectra including but not limited to fluorescein and derivatives, rhodamine and derivatives, cyanine dyes and derivatives, bora-3a,4a-diaza-s-indacene (BODIPY®) and derivatives, pyrene, nanoparticles, or non-fluorescent quenching molecules.
- fluorophores with overlapping emission and excitation spectra including but not limited
- the test compounds is labeled with the acceptor fluorophore. Conversely, if the target nucleic acid is labeled with the acceptor fluorophore, then the test compounds is labeled with the donor fluorophore.
- a wide variety of labels may be used, with the choice of label depending on sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.
- the fluorophore on the target nucleic acid must be in close proximity to the binding site of the test compounds, but should not be incorporated into a target nucleic acid at the specific binding site at which test compounds are likely to bind, since the presence of a covalently attached label might interfere sterically or chemically with the binding of the test compounds at this site.
- HTRF time-resolved fluorescence
- Fluorescence spectroscopy has traditionally been used to characterize DNA- protein and protein-protein interactions, but fluorescence spectroscopy has not been widely used to characterize RNA-protein interactions because of an interfering absorption of RNA nucleotides with the intrinsic tryptophan fluorescence of proteins (Xavier et al, 2000, Trends Biotechnol. 18(8):349-356.). However, fluorescence spectroscopy has been used in studying the single tryptophan residue within the arginine-rich RNA-binding domain of Rev protein and its interaction with the RRE in a time-resolved fluorescence study (Kwon & Carson, 1998, Anal. Biochem. 264:133-140). Thus, in this invention, fluorescence spectroscopy is less preferred if the test compounds or peptides or proteins possess intrinsic tryptophan fluorescence. However, fluorescence spectroscopy can be used for test compounds that do not possess intrinsic fluorescence.
- SPR Surface plasmon resonance
- the evanescent wave profile depends astonishingly on the refractive index of the medium it probes.
- the angle at which absorption occurs is very sensitive to the refractive changes in the external medium.
- All proteins and nucleic acids are known to change the refractive index of water by a similar amount per unit mass, irrespective of their amino acid or nucleotide composition (the refractive index change is different for proteins and nucleic acids).
- the refractive index change is different for proteins and nucleic acids.
- one member of a complex is immobilized in a dextran layer and then the other member is introduced into the solution, either in a flow cell (Biacore AB, Uppsala, Sweden) or a stirred cuvette (Affinity Sensors, Santa Fe, New Mexico). It has been determined that there is a linear correlation between the surface concentration of protein or nucleic acid and the shift in resonance angle, which can be used to quantitate kinetic rate constants and/or the equilibrium constants.
- the target RNA may be immobilized to the sensor surface through a streptavidin-biotin linkage, the linkage of which is disclosed by Crouch et al. (Methods Mol. Biol, 1999, 118:143-160).
- the RNA is biotinylated either during synthesis or post-synthetically via the conversion of the 3' terminal ribonucleoside of the RNA into a reactive free amino group or using a T7 polymerase incorporated guanosine monophosphorothioate at the 5' end.
- SPR has been used to determine the stoichiometry and affinity of the interaction between the HIV Rev protein and the RRE (Van Ryk & Venkatesan, 1999, J. Biol.
- the target nucleic acid can be immobilized to a sensor surface (e.g., by a streptavidin-biotin linkage) and SPR can be used to (a) determine whether the target RNA binds a test compound and (b) further characterize the binding of the target nucleic acids of the present invention to a test compound.
- a sensor surface e.g., by a streptavidin-biotin linkage
- SPR can be used to (a) determine whether the target RNA binds a test compound and (b) further characterize the binding of the target nucleic acids of the present invention to a test compound.
- the mass spectrometer operation on the material to be analyzed is repeated a fixed number of times and the stored control sample values at each m/z ratio level at each time increment are subtracted from each corresponding one from the operational runs, thus producing a difference value at each mass ratio for each of the multiple runs at each time increment. If the MS value minus the background noise does not exceed a preset value, the m/z ratio data point is not recorded, thus eliminating background noise, chemical noise and false positive peaks from the mass spectrometer data.
- the stored data for each of the multiple runs is then compared to a predetermined value at each m/z ratio and the resultant series of peaks, which are now determined to be above the background, is stored in the m/z points in which the peaks are of significance.
- the target nucleic acid complexed to a test compound can be determined by any of the mass spectrometry processed described supra. Furthermore, mass spectrometry can also be used to elucidate the structure of the test compound.
- SPA Scintillation Proximity Assay
- one embodiment of the present invention comprises (a) labeling of the target nucleic acid with a radioactive or fluorescent label; (b) contacted the labeled nucleic acid with test compounds, wherein each test compound is in a microtiter well coated with scintillant and is tethered to the microtiter well; and (c) identifying and quantifying the test compounds bound to the target nucleic acid with SPA, wherein the test compound is identified by virtue of its location in the microplate.
- NMR spectroscopy is a valuable technique for identifying complexed target nucleic acids by qualitatively determining changes in chemical shift, specifically from distances measured using relaxation effects, and NMR-based approaches have been used in the identification of small molecule binders of protein drug targets (Xavier et al, 2000, Trends Biotechnol. 18(8):349-356).
- SAR structure-activity relationships
- the signal from the bound molecule is monitored by employing line broadening, transferred NOEs and pulsed field gradient diffusion measurements (Moore, 1999, Curr. Opin. Biotechnol. 10:54-58).
- a strategy for lead generation by NMR using a library of small molecules has been recently described (Fejzo et al, 1999, Chem. Biol. 6:755-769).
- the target nucleic acid complexed to a test compound can be determined by SAR by NMR. Furthermore, SAR by NMR can also be used to elucidate the structure of the test compound.
- size-exclusion chromatography is used to purify test compounds that are bound to a target nucleic from a complex mixture of compounds.
- Size-exclusion chromatography separates molecules based on their size and uses gel-based media comprised of beads with specific size distributions. When applied to a column, this media settles into a tightly packed matrix and forms a complex array of pores. Separation is accomplished by the inclusion or exclusion of molecules by these pores based on molecular size. Small molecules are included into the pores and, consequently, their migration through the matrix is retarded due to the added distance they must travel before elution. Large molecules are excluded from the pores and migrate with the void volume when applied to the matrix.
- a target nucleic acid is incubated with a mixture of test compounds while free in solution and allowed to reach equilibrium.
- test compounds free in solution are retained by the column, and test compounds bound to the target nucleic acid are passed through the column.
- spin columns commonly used for "desalting" of nucleic acids will be employed to separate bound from unbound test compounds (e.g., Bio-Spin columns manufactured by BIO-RAD).
- the size exclusion matrix is packed into multiwell plates to allow high throughput separation of mixtures (e.g., PLASMID 96-well SEC plates manufactured by Millipore).
- affinity capture is used to purify test compounds that are bound to a target nucleic acid labeled with an affinity tag from a complex mixture of compounds.
- a target nucleic acid labeled with an affinity tag is incubated with a mixture of test compounds while free in solution and then captured to a solid support once equilibrium has been established; alternatively, target nucleic acids labeled with an affinity tag can be captured to a solid support first and then allowed to reach equilibrium with a mixture of test compounds.
- the solid support is typically comprised of, but not limited to, cross-linked agarose beads that are coupled with a ligand for the affinity tag.
- the solid support may be a glass, silicon, metal, or carbon, plastic (polystyrene, polypropylene) surface with or without a self-assembled monolayer (SAM) either with a covalently attached ligand for the affinity tag, or with inherent affinity for the tag on the target nucleic acid.
- plastic polystyrene, polypropylene
- SAM self-assembled monolayer
- retention of bound compounds and removal of unbound compounds is facilitated by washing the solid support with large excesses of binding reaction buffer.
- retention of high affinity compounds and removal of low affinity compounds can be accomplished by a number of means that increase the stringency of washing; these means include, but are not limited to, increasing the number and duration of washes, raising the salt concentration of the wash buffer, addition of detergent or surfactant to the wash buffer, and addition of non-specific competitor to the wash buffer.
- the test compounds themselves are detectably labeled with fluorescent dyes, radioactive isotopes, or nanoparticles.
- fluorescent dyes e.g., fluorescent dyes, radioactive isotopes, or nanoparticles.
- binding between the test compounds and the target nucleic acid can be determined by the presence of the detectable label on the test compound using fluorescence.
- bound compounds with high affinity for the target nucleic acid can be eluted from the immobilized target nucleic acids and analyzed.
- the elution of test compounds can be accomplished by any means that break the non-covalent interactions between the target nucleic acid and compound.
- Means for elution include, but are not limited to, changing the pH, changing the salt concentration, the application of organic solvents, and the application of molecules that compete with the bound ligand.
- the means employed for elution will release the compound from the target RNA, but will not effect the interaction between the affinity tag and the solid support, thereby achieving selective elution of test compound.
- a preferred embodiment will employ an elution buffer that is volatile to allow for subsequent concentration by lyophilization of the eluted compound (e.g., 0 M to 5 M ammonium acetate). 5.5.9. Nanoparticle Aggregation
- both the target nucleic acid and the test compounds are labeled with nanoparticles.
- a nanoparticle is a cluster of ions with controlled size from 0.1 to 1000 nm comprised of metals, metal oxides, or semiconductors including, but not limited to Ag 2 S, ZnS, CdS, CdTe, Au, or TiO 2 .
- Methods for the attachment of nucleic acids and small molecules to nanoparticles are well know to one of skill in the art (reviewed in Niemeyer, 2001, Angew. Chem. Int. Ed. 40:4129-4158. The references cited therein are hereby incorporated by reference in their entireties).
- test compound can be deconvoluted, e.g., by cross-referencing the positive sample to original compound list that was applied to the individual test assays.
- the sequence of the test compound can be determined by direct sequencing of the peptide or nucleic acid. Such methods are well known to one of skill in the art.
- a number of physico-chemical techniques can be used for the de novo characterization of test compounds bound to the target.
- Mass spectrometry e.g., electrospray ionization (“ESI”) and matrix-assisted laser desorption-ionization (“MALDI”), Fourier-transform ion cyclotron resonance (“FT- ICR”)
- ESI electrospray ionization
- MALDI matrix-assisted laser desorption-ionization
- FT- ICR Fourier-transform ion cyclotron resonance
- mass spectroscopy is that separation of a bound and unbound complex and test compound structure elucidation can be carried out in a single step.
- MALDI uses a pulsed laser for desorption of the ions and a time-of-flight analyzer, and has been used for the detection of noncovalent tRNA:amino-acyl-tRNA synthetase complexes (Gruic-Sovulj et al, 1997, J. Biol. Chem. 272:32084-32091).
- covalent cross-linking between the target nucleic acid and the test compound is required for detection, since a non-covalently bound complex may dissociate during the MALDI process.
- ESI mass spectrometry (“ESI-MS”) has been of greater utility for studying non-covalent molecular interactions because, unlike the MALDI process, ESI-MS generates molecular ions with little to no fragmentation (Xavier et al, 2000, Trends Biotechnol. 18(8):349-356). ESI-MS has been used to study the complexes formed by HIV Tat peptide and protein with the TAR RNA (Sannes-Lowery et al, 1997, Anal. Chem. 69:5130-5135).
- FT-ICR Fourier-transform ion cyclotron resonance
- An advantage of mass spectroscopy is not only the elucidation of the structure of the test compound, but also the determination of the structure of the test compound bound to the preselected target RNA. Such information can enable the discovery of a consensus structure of a test compound that specifically binds to a preselected target RNA.
- NMR spectroscopy is a technique for identifying binding sites in target nucleic acids by qualitatively determining changes in chemical shift, specifically from distances measured using relaxation effects.
- Examples of NMR that can be used for the invention include, but are not limited to, one-dimentional NMR, two- dimentional NMR, correlation spectroscopy ("COSY”), and nuclear Overhauser effect (“NOE”) spectroscopy.
- COSY correlation spectroscopy
- NOE nuclear Overhauser effect
- an advantage of NMR is the not only the elucidation of the structure of the test compound, but also the determination of the structure of the test compound bound to the preselected target RNA. Such information can enable the discovery of a consensus structure of a test compound that specifically binds to a preselected target RNA.
- Vibrational spectroscopy e.g. infrared (IR) spectroscopy or Raman spectroscopy
- IR infrared
- Raman spectroscopy can be used for elucidating the structure of the test compound on the isolated bead.
- Infrared spectroscopy measures the frequencies of infrared light (wavelengths from 100 to 10,000 nm) absorbed by the test compound as a result of excitation of vibrational modes according to quantum mechanical selection rules which require that absorption of light cause a change in the electric dipole moment of the molecule.
- the infrared spectrum of any molecule is a unique pattern of absorption wavelengths of varying intensity that can be considered as a molecular fingerprint to identify any compound.
- Infrared spectra can be measured in a scanning mode by measuring the absorption of individual frequencies of light, produced by a grating which separates frequencies from a mixed-frequency infrared light source, by the test compound relative to a standard intensity (double-beam instrument) or pre-measured ('blank') intensity (single-beam instrument).
- infrared spectra are measured in a pulsed mode (FT-IR) where a mixed beam, produced by an interferometer, of all infrared light frequencies is passed through or reflected off the test compound.
- FT-IR pulsed mode
- the resulting interferogram which may or may not be added with the resulting interferograms from subsequent pulses to increase the signal strength while averaging random noise in the electronic signal, is mathematically transformed into a spectrum using Fourier Transform or Fast Fourier Transform algorithms.
- Raman spectroscopy measures the difference in frequency due to absorption of infrared frequencies of scattered visible or ultraviolet light relative to the incident beam.
- the incident monochromatic light beam usually a single laser frequency, is not truly absorbed by the test compound but interacts with the electric field transiently. Most of the light scattered off the sample with be unchanged (Rayleigh scattering) but a portion of the scatter light will have frequencies that are the sum or difference of the incident and molecular vibrational frequencies.
- the selection rules for Raman (inelastic) scattering require a change in polarizability of the molecule. While some vibrational transitions are observable in both infrared and Raman spectrometry, must are observable only with one or the other technique.
- the Raman spectrum of any molecule is a unique pattern of absorption wavelengths of varying intensity that can be considered as a molecular fingerprint to identify any compound.
- Raman spectra are measured by submitting monochromatic light to the sample, either passed through or preferably reflected off, filtering the Rayleigh scattered light, and detecting the frequency of the Raman scattered light.
- An improved Raman spectrometer is described in US Patent No. 5,786,893 to Fink et al, which is hereby incorporated by reference.
- Vibrational microscopy can be measured in a spatially resolved fashion to address single beads by integration of a visible microscope and spectrometer.
- a microscopic infrared spectrometer is described in U.S. Patent No. 5,581,085 to Refiner et al. , which is hereby incorporated by reference in its entirety.
- An instrument that simultaneously performs a microscopic infrared and microscopic Raman analysis on a sample is described in U.S. Patent No. 5,841,139 to Sostek et al, which is hereby incorporated by reference in its entirety.
- test compounds can be identified by matching the IR or Raman spectra of a test compound to a dataset of vibrational (IR or Raman) spectra previously acquired for each compound in the combinatorial library.
- IR or Raman vibrational
- test compounds identified in the binding assay can be tested for biological activity using host cells containing or engineered to contain the target RNA element coupled to a functional readout system.
- the lead compound can be tested in a host cell engineered to contain the target RNA element controlling the expression of a reporter gene.
- the lead compounds are assayed in the presence or absence of the target RNA.
- a phenotypic or physiological readout can be used to assess activity of the target RNA in the presence and absence of the lead compound.
- the lead compound can be tested in a host cell engineered to contain the target RNA element controlling the expression of a reporter gene, such as, but not limited to, ⁇ -galactosidase, green fluorescent protein, red fluorescent protein, luciferase, chloramphenicol acetyltransferase, alkaline phosphatase, and ⁇ - lactamase.
- a reporter gene such as, but not limited to, ⁇ -galactosidase, green fluorescent protein, red fluorescent protein, luciferase, chloramphenicol acetyltransferase, alkaline phosphatase, and ⁇ - lactamase.
- a cDNA encoding the target element is fused upstream to a reporter gene wherein translation of the reporter gene is repressed upon binding of the lead compound to the target RNA. In other words, the steric hindrance caused by the binding of the lead compound to the target RNA repressed the translation of the reporter gene.
- a phenotypic or physiological readout can be used to assess activity of the target RNA in the presence and absence of the lead compound.
- the target RNA may be overexpressed in a cell in which the target RNA is endogenously expressed.
- the in vivo effect of the lead compound can be assayed by measuring the cell growth or viability of the target cell.
- a reporter gene can also be fused downstream of the target RNA sequence and the effect of the lead compound on reporter gene expression can be assayed.
- the lead compounds identified in the binding assay can be tested for biological activity using animal models for a disease, condition, or syndrome of interest. These include animals engineered to contain the target RNA element coupled to a functional readout system, such as a transgenic mouse. Animal model systems can also be used to demonstrate safety and efficacy.
- Compounds displaying the desired biological activity can be considered to be lead compounds, and will be used in the design of congeners or analogs possessing useful pharmacological activity and physiological profiles.
- molecular modeling techniques can be employed, which have proven to be useful in conjunction with synthetic efforts, to design variants of the lead that can be more effective. These applications may include, but are not limited to, Pharmacophore Modeling (cf Lamothe, et al.1997, J. Med. Chem. 40: 3542; Housing et al. 1996, J. Med. Chem. 39: 285; Beusen etal. 1995, Biopolymers 36: 181; P. Fossa et al. 1998, Comput. Aided Mol. Des.
- RNA structural programs including, but not limited to mFold (as described by Zuker et al. Algorithms and Thermodynamics for RNA Secondary Structure Prediction: A Practical Guide in RNA Biochemistry and Biotechnology pp. 11-43, J. Barciszewski & B.F.C. Clark, eds. (NATO ASI Series, Kluwer Academic Publishers, 1999) and Mathews et al. 1999 J. Mol. Biol.
- RNAmotif Macke et al 2001, Nucleic Acids Res. 29: 4724-4735; and the Vienna RNA package (Hofacker et al. 1994, Monatsh. Chem. 125: 167-188).
- Molecular modeling tools employed may include those from Tripos, Inc., St. Louis, Missouri (e.g., Sybyl/UNITY, CONCORD, DiverseSolutions), Accelerys, San Diego, California (e.g., Catalyst, Wisconsin Package ⁇ BLAST, etc. ⁇ ), Schrodinger, Portland, Oregon (e.g., QikProp, QikFit, Jaguar) or other such vendors as BioDesign, Inc. (Pasadena, California), Allelix, Inc. (Mississauga, Ontario, Canada), and Hypercube, Inc. (Cambridge, Ontario, Canada), and may include privately designed and/or "academic" software (e.g. RNAMotif, mFOLD).
- QSARs Quantitative Structural Activity Relationships
- Biologically active compounds identified using the methods of the invention or a pharmaceutically acceptable salt thereof can be administered to a patient, preferably a mammal, more preferably a human, suffering from a disease whose progression is associated with a target RNA:host cell factor interaction in vivo.
- such compounds or a pharmaceutically acceptable salt thereof is administered to a patient, preferably a mammal, more preferably a human, as a preventative measure against a disease associated with an RNA:host cell factor interaction in vivo.
- treatment refers to an amelioration of a disease, or at least one discernible symptom thereof.
- treatment or “treating” refers to an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient.
- treatment or “treating” refers to inhibiting the progression of a disease, either physically, e.g., stabilization of a discernible symptom, physiologically, e.g., stabilization of a physical parameter, or both.
- treatment or “treating” refers to delaying the onset of a disease.
- the compound or a pharmaceutically acceptable salt thereof is administered to a patient, preferably a mammal, more preferably a human, as a preventative measure against a disease associated with an RNA:host cell factor interaction in vivo.
- prevention or “preventing” refers to a reduction of the risk of acquiring a disease.
- the compound or a pharmaceutically acceptable salt thereof is administered as a preventative measure to a patient.
- the patient can have a genetic predisposition to a disease, such as a family history of the disease, or a non-genetic predisposition to the disease. Accordingly, the compound and pharmaceutically acceptable salts thereof can be used for the treatment of one manifestation of a disease and prevention of another.
- the compound or a pharmaceutically acceptable salt thereof is preferably administered as component of a composition that optionally comprises a pharmaceutically acceptable vehicle.
- the composition can be administered orally, or by any other convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal, and intestinal mucosa, etc.) and may be administered together with another biologically active agent. Administration can be systemic or local.
- Various delivery systems are known, e.g., encapsulation in liposomes, microparticles, microcapsules, capsules, etc., and can be used to administer the compound and pharmaceutically acceptable salts thereof.
- Methods of administration include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectally, by inhalation, or topically, particularly to the ears, nose, eyes, or skin.
- the mode of administration is left to the discretion of the practitioner. In most instances, administration will result in the release of the compound or a pharmaceutically acceptable salt thereof into the bloodstream. hi specific embodiments, it may be desirable to administer the compound or a pharmaceutically acceptable salt thereof locally.
- This may be achieved, for example, and not by way of limitation, by local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non- porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
- Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir.
- Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent, or via perfusion in a fluorocarbon or synthetic pulmonary surfactant.
- the compound and pharmaceutically acceptable salts thereof can be formulated as a suppository, with traditional binders and vehicles such as triglycerides.
- the compound and pharmaceutically acceptable salts thereof can be delivered in a vesicle, in particular a liposome (see Langer, 1990, Science 249:1527-1533; Treat et al, in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).
- a liposome see Langer, 1990, Science 249:1527-1533; Treat et al, in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).
- the compound and pharmaceutically acceptable salts thereof can be delivered in a controlled release system (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
- a controlled release system See, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
- Other controlled- release systems discussed in the review by Langer, 1990, Science 249:1527-1533
- a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al, 1980, Surgery 88:507 Saudek et al, 1989, N. Engl. J. Med. 321 :574).
- polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al, 1985, Science 228:190; During et al, 1989, Ann. Neurol. 25:351; Howard et al, 1989, J. Neurosurg. 71 : 105).
- a controlled-release system can be placed in proximity of a target RNA of the compound or a pharmaceutically acceptable salt thereof, thus requiring only a fraction of the systemic dose.
- compositions comprising the compound or a pharmaceutically acceptable salt thereof (“compound compositions”) can additionally comprise a suitable amount of a pharmaceutically acceptable vehicle so as to provide the form for proper administration to the patient.
- the term "pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, mammals, and more particularly in humans.
- vehicle refers to a diluent, adjuvant, excipient, or carrier with which a compound of the invention is administered.
- Such pharmaceutical vehicles can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
- the pharmaceutical vehicles can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like.
- auxiliary, stabilizing, thickening, lubricating and coloring agents may be used.
- the pharmaceutically acceptable vehicles are preferably sterile. Water is a preferred vehicle when the compound of the invention is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid vehicles, particularly for injectable solutions.
- Suitable pharmaceutical vehicles also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
- excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
- Compound compositions if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
- Compound compositions can take the form of solutions, suspensions, emulsion, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained- release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use.
- the pharmaceutically acceptable vehicle is a capsule (see e.g., U.S. Patent No. 5,698,155).
- suitable pharmaceutical vehicles are described in Remington's Pharmaceutical Sciences, Alfonso R. Gennaro, ed., Mack Publishing Co. Easton, PA, 19th ed., 1995, pp. 1447 to 1676, incorporated herein by reference.
- compositions for oral delivery may be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example.
- Orally administered compositions may contain one or more agents, for example, sweetening agents. such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of ⁇ vintergreen, or cherry; coloring agents; and preserving agents, to provide a pharmaceutically palatable preparation.
- compositions can be coated to delay disintegration and absorption in the gastrointestinal tract thereby providing a sustained action over an extended period of time.
- Selectively permeable membranes surrounding an osmotically active driving compound are also suitable for orally administered compositions.
- fluid from the environment surrounding the capsule is imbibed by the driving compound, which swells to displace the agent or agent composition through an aperture.
- delivery platforms can provide an essentially zero order delivery profile as opposed to the spiked profiles of immediate release formulations.
- a time delay material such as glycerol monostearate or glycerol stearate may also be used.
- compositions can include standard vehicles such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Such vehicles are preferably of pharmaceutical grade.
- compositions for intravenous administration comprise sterile isotonic aqueous buffer. Where necessary, the compositions may also include a solubilizing agent.
- the compound or a pharmaceutically acceptable salt thereof can be formulated for intravenous administration.
- Compositions for intravenous administration may optionally include a local anesthetic such as lignocaine to lessen pain at the site of the injection.
- the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
- the compound or a pharmaceutically acceptable salt thereof is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline.
- an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
- the amount of a compound or a pharmaceutically acceptable salt thereof that will be effective in the treatment of a particular disease will depend on the nature of the disease, and can be determined by standard clinical techniques.
- in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges.
- the precise dose to be employed will also depend on the route of administration, and the seriousness of the disease, and should be decided according to the judgment of the practitioner and each patient's circumstances.
- suitable dosage ranges for oral administration are generally about 0.001 milligram to about 200 milligrams of a compound or a pharmaceutically acceptable salt thereof per kilogram body weight per day.
- the oral dose is about 0.01 milligram to about 100 milligrams per kilogram body weight per day, more preferably about 0.1 milligram to about 75 milligrams per kilogram body weight per day, more preferably about 0.5 milligram to 5 milligrams per kilogram body weight per day.
- the dosage amounts described herein refer to total amounts administered; that is, if more than one compound is administered, or if a compound is administered with a therapeutic agent, then the preferred dosages correspond to the total amount administered.
- Oral compositions preferably contain about 10% to about 95% active ingredient by weight.
- Suitable dosage ranges for intravenous (i.v.) administration are about 0.01 milligram to about 100 milligrams per kilogram body weight per day, about 0.1 milligram to about 35 milligrams per kilogram body weight per day, and about 1 milligram to about 10 milligrams per kilogram body weight per day.
- Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight per day to about 1 mg/kg body weight per day.
- Suppositories generally contain about 0.01 milligram to about 50 milligrams of a compound of the invention per kilogram body weight per day and comprise active ingredient in the range of about 0.5% to about 10% by weight.
- Suitable dosages for intradermal, intramuscular, intraperitoneal, subcutaneous, epidural, sublingual, intracerebral, intravaginal, transdermal administration or administration by inhalation are in the range of about 0.001 milligram to about 200 milligrams per kilogram of body weight per day.
- Suitable doses for topical administration are in the range of about 0.001 milligram to about 1 milligram, depending on the area of administration.
- Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. Such animal models and systems are well known in the art.
- the compound and pharmaceutically acceptable salts thereof are preferably assayed in vitro and in vivo, for the desired therapeutic or prophylactic activity, prior to use in humans.
- in vitro assays can be used to determine whether it is preferable to administer the compound, a pharmaceutically acceptable salt thereof, and/or another therapeutic agent.
- Animal model systems can be used to demonstrate safety and efficacy.
- a variety of compounds can be used for treating or preventing diseases in mammals.
- Types of compounds include, but are not limited to, peptides, peptide analogs including peptides comprising non-natural amino acids, e.g., D-amino acids, phosphorous analogs of amino acids, such as ⁇ -amino phosphonic acids and ⁇ -amino phosphinic acids, or amino acids having non-peptide linkages, nucleic acids, nucleic acid analogs such as phosphorothioates or peptide nucleic acids (“PNAs”), hormones, antigens, synthetic or
- the therapeutic targets presented herein are by way of example, and the present invention is not to be limited by the targets described herein.
- the therapeutic targets presented herein as DNA sequences are understood by one of skill in the art that the sequences can be converted to RNA sequences.
- TNF- ⁇ Tumor Necrosis Factor Alpha
- Group I AU-Rich Element (ARE) Cluster in 3' untranslated region 5' AUUUAUUUAUUUAUUUAUUUA 3' (SEQ ID NO: 1)
- GM-CSF Granulocyte-macrophage Colony Stimulating Factor
- GenBank Accession # XM_003751 GenBank Accession # XM_003751 :
- Group I AU-Rich Element (ARE) Cluster in 3' untranslated region 5' AUUUAUUUAUUUAUUUAUUUA 3' (SEQ ID NO: 1)
- IL-6 Interleukin 6
- VEGF Vascular Endothelial Growth Factor
- HIV-1 Human Immunodeficiency Virus I
- HCV Hepatitis C Virus
- XIAP X-linked Inhibitor of Apoptosis Protein
- TK buffer is composed of 50 mM Tris-HCI pH
- Tris-borate-EDTA (TBE) buffer is composed of 45 mM Tris-borate pH 8.0, and 1 mM EDTA.
- Tris-Potassium chloride- magnesium (TKM) buffer is composed of 50 mM Tris-HCI pH 7.4, 20mM KCl, 0.1%Triton X-100 and 5mM MgCl 2 .
- RNA oligonucleotides were purchased from Dharmacon, Inc, Lafayette,
- RNA ligase (NEBiolabs) in 10% DMSO as per manufacturer's instructions.
- the labeled oligonucleotides were purified using G-25 Sephadex columns (Boehringer Mannheim).
- Tat peptide (YGRKKRRQRRRP (SEQ ID NO: 31); single letter amino acid code).
- RNA-small molecule interactions with natural occurring RNA structures such as ribosomes
- chemical footprinting or toe printing Moazed & Noller, 1987, Nature 327:389-394; Woodcock et al, 1991, EMBO J. 10:3099-3103; Yoshizawa et al, 1998, EMBO J. 17:6437-6448.
- gel mobility shift assays to monitor RNA-small molecule interactions are described.
- RNA oligonucleotide corresponding to the well- characterized gentamicin binding site on the 16S rRNA (Moazed & Noller, 1987, Nature 327:389-394) and the equally well-characterized EDV-l TAT protein binding site on the HIV-1 TAR element (Huq et al, 1999, Nucleic Acids Res. 27: 1084-1093) were chosen.
- the purpose of these experiments is to lay the groundwork for the use of chromatographic techniques in a high throughput fashion, such as microcapillary electrophoresis, for drug discovery.
- a gel retardation assay was performed using the Tat 47 . 58 peptide and the TAR RNA oligonucleotide. As shown in Figure 1, in the presence of the Tat peptide, a clear shift is visible when the products are separated on a 12% non-denaturing polyacrylamide gel. In the reaction that lacks peptide, only the free RNA is visible. These observations confirm previous reports made using other Tat peptides (Hamy et al, 1997, Proc. Natl. Acad. Sci. USA 94:3548-3553; Huq et al, 1999, Nucleic Acids Res. 27: 1084- 1093).
- the temperature of the reaction when gentamicin is added is also important.
- gentamicin is present in the reaction during the entire denaturation/renaturation cycle, that is, when gentamicin is added at 90C°C or 85 °C, a gel shift is visualized (data not shown).
- gentamicin is added after the renaturation step has proceeded to 75 °C, a mobility shift is not produced.
- gentamicin may recognize and interact with an RNA structure formed early in the renaturation process.
- results presented in this Example indicate that interactions between a peptide and its target RNA, such as the Tat peptide and TAR RNA, can be monitored by gel retardation assays in an automated capillary electrophoresis system.
- Tris-potassium chloride (TK) buffer is composed of 50 mM Tris-HCI pH 7.4, 20mM KCl, 0.1%Triton X-100, and 0.5mM MgCl 2 .
- Tris-borate-EDTA (TBE) buffer is
- Tris-Potassium chloride- magnesium (TKM) buffer is composed of 50 mM Tris-HCI pH 7.4, 20mM KCl, 0.1%Triton X-100 and 5mM MgCl 2 .
- RNA oligonucleotides were purchased from Dharmacon, Inc, Lafayette, CO). 500 pmole of a 5' fluorescein labeled oligonucleotide corresponding to the HIV-1 TAR element TAR RNA (5'-GGCGUCACACCUUCGGGUGAAGUCGCC-3' (SEQ ID NO: 30); Huq et al, 1999, Nucleic Acids Research. 27:1084-1093; Hwang et al, 1999, Proc. Natl. Acad. Sci. USA 96:12997-13002) was used.
- Tat-TAR gel retardation 5' fluorescein labeled oligonucleotide corresponding to the HIV-1 TAR element TAR RNA
- a method for identifying a test compound that binds to a target RNA molecule comprising the steps of (a) contacting a detectably labeled target RNA molecule with a library of test compounds under conditions that permit direct binding of the labeled target RNA to a member of the library of test compounds so that a detectably labeled target RNA:test compound complex is formed; (b) separating the detectably labeled target RNA:test compound complex formed in step(a) from uncomplexed target RNA molecules and test compounds; and (c) determining a structure of the test compound bound to the RNA in the RNA:test compound complex.
- the target RNA molecule contains an HIV TAR element, internal ribosome entry site, "slippery site”, instability element, or adenylate uridylate-rich element.
- RNA molecule is an element derived from the mRNA for tumor necrosis factor alpha ("TNF- ⁇ ”), granulocyte-though macrophage colony stimulating factor (“GM-CSF”), interleukin 2 (“IL-2”), interleukin 6 (“IL-6”), vascular endothelial growth factor (“VEGF”), human immunodeficiency virus I (“HIV-1”), hepatitis C virus (“HCV” - genotypes la & lb), ribonuclease P RNA (“RNaseP”), X-linked inhibitor of apoptosis protein (“XIAP”), or survivin.
- TNF- ⁇ tumor necrosis factor alpha
- GM-CSF macrophage colony stimulating factor
- IL-2 interleukin 2
- IL-6 interleukin 6
- VEGF vascular endothelial growth factor
- HCV-1 human immunodeficiency virus I
- HCV hepatitis C virus
- RNaseP ribon
- RNA is labeled with a fluorescent dye, phosphorescent dye, ultraviolet dye, infrared dye, visible dye, radiolabel, enzyme, spectroscopic colorimetric label, affinity tag, or nanoparticle.
- a combinatorial library comprising peptoids; random bio-oligomers; diversomers such as hydantoins, benzodiazepines and dipeptides; vinylogous polypeptides; nonpeptidal peptidomimetics; oligocarbamates; peptidyl phosphonates; peptide nucleic acid libraries; antibody libraries; carbohydrate libraries; and small organic molecule libraries, including but not limited to, libraries of benzodiazepines, isoprenoids, thiazolidinones, strictly - metathiazanones, pyrrolidines, morpholino compounds, or diazepindiones. 6.
- screening a library of test compounds comprises contacting the test compound with the target nucleic acid in the presence of an aqueous solution, the aqueous solution comprising a buffer and a combination of salts, preferably approximating or mimicking physiologic conditions.
- aqueous solution optionally further comprises non-specific nucleic acids comprising DNA, yeast tRNA, salmon sperm DNA, homoribopolymers, and nonspecific RNAs.
- the aqueous solution further comprises a buffer, a combination of salts, and optionally, a detergent or a surfactant.
- the aqueous solution further comprises a combination of salts, from about 0 mM to about 100 mM KCl, from about 0 mM to about 1 M NaCl, and from about 0 mM to about 200 mM MgCl 2 .
- the combination of salts is about 100 mM KCl, 500 mM NaCl, and 10 mM MgCl 2 .
- the solution optionally comprises from about 0.01% to about 0.5% (w/v) of a detergent or a surfactant.
- Any method that detects an altered physical property of a target nucleic acid complexed to a test compound from the unbound target nucleic acid may be used for separation of the complexed and non-complexed target nucleic acids in the method of paragraph 1.
- electrophoresis is used for separation of the complexed and non-complexed target nucleic acids.
- the electrophoresis is capillary electrophoresis.
- fluorescence spectroscopy surface plasmon resonance, mass spectrometry, scintillation, proximity assay, structure-activity relationships ("SAR") by NMR spectroscopy, size exclusion chromatography, affinity chromatography, and nanoparticle aggregation are used for the separation of the complexed and non-complexed target nucleic acids.
- SAR structure-activity relationships
- test compound of the RNA:test compound complex of paragraph 1 is determined, in part, by the type of library of test compounds.
- the combinatorial libraries are small organic molecule libraries, mass spectroscopy, NMR, or vibration spectroscopy are used to determine the structure of the test compounds.
Abstract
Description
Claims
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CA002443711A CA2443711A1 (en) | 2001-04-11 | 2002-04-11 | Methods for identifying small molecules that bind specific rna structural motifs |
JP2002581693A JP2004537037A (en) | 2001-04-11 | 2002-04-11 | Methods for identifying small molecules that bind to specific RNA structural motifs |
EP02725663A EP1377684A4 (en) | 2001-04-11 | 2002-04-11 | Methods for identifying small molecules that bind specific rna structural motifs |
US10/475,024 US20040219545A1 (en) | 2001-04-13 | 2002-04-11 | Methods for identifying small molecules that bind specific rna structural motifs |
US11/347,748 US20060228730A1 (en) | 2001-04-11 | 2006-02-03 | Methods for identifying small molecules that bind specific RNA structural motifs |
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Cited By (13)
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WO2004087884A3 (en) * | 2003-03-27 | 2005-04-14 | Ptc Therapeutics Inc | TARGETING ENZYMES OF THE tRNA SPLICING PATHWAY FOR IDENTIFICATION OF ANTI-FUNGAL AND/OR ANTI-PROLIFERATIVE MOLECULES |
WO2004087070A3 (en) * | 2003-03-27 | 2005-08-04 | Ptc Therapeutics Inc | METHODS OF IDENTIFYING COMPOUNDS THAT TARGET tRNA SPLICING ENDONUCLEASE AND USES OF SAID COMPOUNDS AS ANTI-FUNGAL AGENTS |
WO2004087069A3 (en) * | 2003-03-27 | 2005-08-25 | Ptc Therapeutics Inc | METHODS OF IDENTIFYING COMPOUNDS THAT TARGET tRNA SPLICING ENDONUCLEASE AND USES OF SAID COMPOUNDS AS ANTI-PROLIFERATIVE AGENTS |
JPWO2006054788A1 (en) * | 2004-11-19 | 2008-06-05 | 武田薬品工業株式会社 | Method for screening compounds that control translation of specific mRNA |
US7927791B2 (en) | 2002-07-24 | 2011-04-19 | Ptc Therapeutics, Inc. | Methods for identifying small molecules that modulate premature translation termination and nonsense mediated mRNA decay |
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JP6903617B2 (en) * | 2018-09-19 | 2021-07-14 | 株式会社東芝 | How to determine the molecular probe |
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EP1377684A1 (en) | 2004-01-07 |
JP2004537037A (en) | 2004-12-09 |
EP1377684A4 (en) | 2009-03-18 |
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