WO1999050462A1 - Systemes d'inhibition bacteriens a base d'aptameres (abbis) - Google Patents

Systemes d'inhibition bacteriens a base d'aptameres (abbis) Download PDF

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Publication number
WO1999050462A1
WO1999050462A1 PCT/US1999/006466 US9906466W WO9950462A1 WO 1999050462 A1 WO1999050462 A1 WO 1999050462A1 US 9906466 W US9906466 W US 9906466W WO 9950462 A1 WO9950462 A1 WO 9950462A1
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aptamer
target molecule
cell
growth
expression
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PCT/US1999/006466
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English (en)
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John J. Mekalanos
Jonathan Blum
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President And Fellows Of Harvard College
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Priority to AU33633/99A priority Critical patent/AU3363399A/en
Publication of WO1999050462A1 publication Critical patent/WO1999050462A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1075—Isolating an individual clone by screening libraries by coupling phenotype to genotype, not provided for in other groups of this subclass
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the field of the invention is antimicrobial drug discovery.
  • ABS Advanced Bacterial Inhibition System
  • aptamers chimeric proteins
  • ABBIS is useful for defining potentially novel therapeutic targets and for antimicrobial drug screening assays.
  • the invention features a method for identifying an aptamer that inhibits the viability, growth, or virulence of an organism.
  • the method comprises: a) transforming the cells of the organism with an aptamer expression library, wherein expression of aptamers in the library is tightly regulated; b) inducing aptamer expression; c) identifying cells that express aptamers that inhibit the viability, growth, or virulence of the cells, relative to control aptamers that do not inhibit viability, growth, or virulence; and d) isolating aptamer-encoding DNA from cells that express aptamers that inhibit the viability, growth, or virulence of the cells.
  • the invention features a method for identifying an aptamer that inhibits the biological function of an essential target molecule.
  • the method comprises: a) transforming cells expressing the essential target molecule with an aptamer expression library, wherein expression of aptamers -3- in the library is tightly regulated; b) inducing aptamer expression; c) identifying cells that express aptamers that inhibit the viability, growth, or virulence of the cells, relative to control aptamers that do not inhibit viability, growth, or virulence; and d) isolating aptamer-encoding DNA from cells that express aptamers that inhibit the viability, growth, or virulence of the cells.
  • the method further includes sequencing the isolated aptamer- encoding DNA.
  • the invention features a method for identifying an essential target molecule in an organism, comprising: a) identifying an aptamer that inhibits the biological function of an essential target molecule, wherein identification of the aptamer comprises the method of the second aspect of the invention; b) exposing test samples containing potential essential target molecules to the aptamer ; c) assaying for an interaction between an aptamer and an essential target molecule; and d) determining the identity of an essential target molecule that interacts with the aptamer.
  • the essential target molecule may be a protein (e.g., the enzyme Thy A), a carbohydrate, a lipid, or a nucleic acid.
  • the essential target molecule may be identified via its interaction with the inhibitory aptamer through basic biochemical and genetic analyses that are known to those of skill in the art.
  • the invention features a method for producing a drug screening strain.
  • the method comprises: a) transforming cells expressing an essential target molecule with an aptamer expression library, wherein expression of aptamers in the library is tightly regulated; b) inducing aptamer expression; c) identifying cells that express aptamers at levels that partially inhibit the viability, growth, or virulence of the cells; and d) isolating the -4- identified cells for use in drug screens.
  • the drug screening strain is produced by making the cells hypersensitive to drugs using an aptamer that alters the permeability of the cell.
  • step (c) is relative to a cell expressing a control aptamer that does not inhibit viability, growth, or virulence.
  • the invention features a method for producing a drug screening strain.
  • the method comprises: a) producing Thy A/target molecule chimeras by inserting a portion of the target molecule into a permissive location within ThyA; and b) producing cells that express the Thy A/target molecule chimeras.
  • the invention features a method for detecting a compound that inhibits the biological function of a target molecule.
  • the method comprises: a) inducing a cell from step (d) of the fourth aspect of the invention to express the aptamer at subinhibitory levels; b) exposing the cell to a test compound; and c) assaying for decreased cell viability, growth, or virulence, relative to a cell not exposed to the compound.
  • the invention features a method for detecting a compound that inhibits the viability, growth, or virulence of a cell.
  • the method comprises: a) inducing a cell from step (d) of the fourth aspect of the invention to express an aptamer at subinhibitory levels; b) exposing the cell to a test compound; and c) assaying for decreased cell viability, growth, or virulence, relative to a cell not exposed to the compound.
  • the invention features a method for detecting a compound that inhibits the biological function of a target molecule.
  • the said method comprises: a) exposing a cell from step (b) of the fifth aspect of the invention to a test compound; and b) assaying for decreased cell growth on -5- selective minimal medium lacking thymine.
  • the invention features a method for detecting a compound that inhibits the biological function of a target molecule.
  • the method comprises: a) exposing a cell from step (b) of the fifth aspect of the invention to a test compound; and b) assaying for increased growth on selective medium containing trimethoprim.
  • the invention features a method for constructing an aptamer expression library.
  • the method includes: (a) providing a collection of double- stranded random oligonucleotides; (b) providing a plasmid containing a gene encoding a scaffold protein; (c) generating a collection of aptamer- encoding genes by cloning the random oligonucleotides into a permissive site within the gene encoding the scaffold protein, wherein the oligonucleotides are cloned in frame with the coding sequence of the scaffold protein gene, such that each chimeric gene encodes an aptamer, wherein the aptamer consists essentially of a random peptide constrained within a scaffold protein; and (d) ensuring that each aptamer-encoding gene is operably linked to a promoter within an expression vector, wherein the promoter allows tightly regulated expression of the aptamer-encoding gene.
  • the scaffold includes: (a) providing a
  • the invention features a method of identifying a compound that binds to an essential target molecule.
  • the method includes: (a) exposing the essential target molecule to an inhibitory aptamer, wherein the aptamer was previously identified using an aptamer expression library in which aptamer expression is tightly regulated; (b) exposing the essential target molecule to a test compound; and (c) measuring the amount of aptamer that is bound to the essential target molecule after the essential target molecule is -6- exposed to the test compound, wherein a test compound that decreases the amount of aptamer bound to said essential target molecule, relative to the amount of aptamer bound to the identical essential target molecule not exposed to the test compound, and wherein said test compound binds the essential target molecule more strongly than the test compound binds the aptamer, indicates a compound that binds to an essential target molecule.
  • the essential target molecule may be exposed to the aptamer prior to exposing the essential target molecule to the test compound; the essential target molecule may be exposed to the aptamer after exposing the essential target molecule to the test compound; or the essential target molecule may be exposed simultaneously to the aptamer and the test compound.
  • the essential target molecule and the aptamer are located intracellularly.
  • the method may be used to develop small synthetic molecules that are related in structure to a peptide that confers inhibitory activity upon a given aptamer.
  • the invention features an aptamer comprising the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 10.
  • the invention features a nucleic acid sequence encoding an aptamer comprising the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 10.
  • aptamers random combinatorial peptide sequences, some of which may bind to and inhibit the function of target molecules. Aptamers may be expressed intracellularly by cloning their encoding DNA into an expression vector, such as pBMT or pBMML2, which are described herein. Aptamers may be expressed within the context of a scaffold protein, such as thioredoxin or maltose binding protein.
  • Such scaffold proteins target the aptamer to a particular subcellular location, such as the periplasmic space.
  • Aptamers are usually between 8 and 70 amino acids in length, although an aptamer may be as small as 4 amino acids or larger than 70 amino acids.
  • aptamers may be, for example, 4-8, 8-16, 9-13, 14-25, 16-34, 16-52, 16-70, 26-30, 31-35, 36-40, 41-45, or 46-50 amino acids in length, or greater than 70 amino acids in length.
  • toxic aptamer or “inhibitory aptamer” is meant an aptamer that physically interacts with and interferes with the biological function of an essential target molecule, as defined below. Inhibition of viability, growth or virulence may be complete, partial, or conditional, as described below.
  • aptamer expression library is meant a collection of random oligonucleotide sequences that encodes aptamers, cloned into an expression vector that contains a promoter that directs transcription in the cell type in which the library is to be expressed.
  • the promoter is an inducible promoter, such as the araBAD promoter of E. coli, or one of the other inducible promoter systems known for prokaryotic or eukaryotic cells, including tetracyline-inducible, hormone-inducible, metal ion-inducible, or heat shock- inducible promoter systems.
  • the aptamers are expressed within the context of chimeric aptamer/scaffold proteins.
  • Tightly regulated expression or “tight transcriptional control” is meant that an aptamer is expressed only when it is desirable to do so. Tightly regulated expression may be achieved through the use of an inducible promoter, e.g., the E. coli araBAD promoter described herein.
  • promoter is meant a minimal sequence sufficient to direct -8- transcription. Also included are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell type- specific, tissue-specific, temporal-specific, or inducible by external signals or agents.
  • operably linked is meant that a gene and one or more regulatory sequences are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequences.
  • drug screening strain or “hypersensitized strain” is meant a cell that expresses a toxic or inhibitory aptamer that decreases (but does not completely inhibit) the viability, growth or virulence of the cell, relative to an identical cell that does not express the aptamer.
  • expression of the aptamer renders the screening strain hypersensitive to compounds that inhibit cell viability, growth or virulence, since these processes are already partially compromised within the screening strain.
  • Use of a screening strain increases the sensitivity of a drug screening assay.
  • the cells of a screening strain may be derived from a variety of organisms, such as bacteria, fungi, parasites, insects, animals, and plants.
  • essential target molecule or "target molecule” is meant a molecule whose function is required for survival, growth, mitosis/meiosis, or virulence of a cell (e.g, a bacterial, fungal, parasitic, insect, plant, or mammalian cell).
  • a target molecule may also be a molecule whose function is required for the replication, infectivity, or virulence of a virus.
  • a target molecule may be a protein, nucleic acid, lipid, or carbohydrate molecule that interacts with an aptamer in such a way that the biological function of the target molecule is inhibited. Intracellular aptamer/target molecule interactions may be inferred through the detection of a decrease in cell viability, growth or -9- virulence.
  • Biological functions of target molecules may include (but are not limited to) participation in enzymatic reactions such as DNA replication, RNA transcription, protein translation, transport of ions, molecules, or other nutrients, or may include functions such as maintainence of cell structures.
  • Interaction of an aptamer with an essential target molecule may be lethal, i.e., prevent a cell (or virus) from surviving (or replicating), growing, or undergoing mitosis/meiosis.
  • interaction of an aptamer with an essential target molecule may allow survival of a cell (or virus), but result in severely diminished growth or virulence; e.g., interaction of an aptamer with an essential target molecule may prevent a cell or virus from expressing components required for virulence, such as toxins, pili, flagella, or other structures.
  • An essential target molecule also may be conditionally essential, i.e., required for viability, growth or virulence under certain conditions, but not under other conditions.
  • determining the identity of an essential target molecule is meant a process by which the identity of an essential target molecule that interacts with a given aptamer may be ascertained.
  • the target molecule may be purified from cell extracts or expression libraries using an aptamer affinity column, after which the identity of the target molecule may be determined by biochemical analysis (e.g., peptide sequencing), methods of which are known to those skilled in the arts of biochemistry and molecular biology.
  • subinhibitory levels of an aptamer is meant that an inhibitory aptamer only partially inhibits the biological function of the target molecule with which it interacts.
  • subinhibitory levels of an aptamer within a cell allows cell survival, although the viability, growth -10- rate, or virulence may be decreased relative to the same type of cell not expressing the inhibitory aptamer.
  • cells expressing subinhibitory levels of aptamers may be hypersensitized to the toxic or inhibitory effects of a second compound to which the cell may be exposed, and therefore, are useful for drug screens.
  • the subinhibitory level of a given aptamer is intrinsic to the aptamer and the cell in which the aptamer is expressed.
  • an aptamer present at a relatively high concentration may only be subinhibitory (as opposed to strongly inhibitory) because its physical association with its target molecule is relatively weak.
  • subinhibitory levels of an strongly inhibitory aptamer may be achieved by allowing only a low level of aptamer expression.
  • Expose is meant to allow contact between a, cell, lysate or extract derived from a cell, or molecule derived from a cell, and an aptamer or test compound. Exposing may be done intracellularly: for example, exposing potential target molecules to an aptamer to by expressing the aptamer within the cell.
  • binds more strongly is meant that a test compound binds more tightly to an essential target molecule than to an aptamer by at least 10%, preferably by at least 15%, more preferably by at least 20%, still more preferably by at least 30%, yet more preferably by at least 40%, and most preferably by at least 50%.
  • Relative strength of binding may be determined by well known methods, e.g., affinity chromatography, immunoassay, or Scatchard analysis.
  • decrease is meant a lessening of at least 2-fold (preferably, at least 3-fold, more preferably, at least 5-fold, still more preferably, at least 7- fold, and most preferably, at least 10-fold) in the level of viability, growth, or virulence of a cell or microorganism, in the biological activity (e.g., enzymatic -11- activity) of an essential target molecule, or in the interaction of an essential target molecule with an aptamer.
  • biological activity e.g., enzymatic -11- activity
  • test compound is meant a chemical, be it naturally occurring or artificially derived, that is surveyed for its ability to partially or completely inhibit the activity of a target molecule such that the viability, growth, or virulence of a cell is partially or completely inhibited.
  • Test compounds may include, for example, peptides, polypeptides, synthesized organic molecules, naturally occurring organic molecules, nucleic acid molecules, carbohydrates, and components thereof.
  • assaying is meant analyzing the effect of a treatment administered to cells.
  • the treatment may be, for example, induction of aptamer expression or exposure to a test compound.
  • the analysis may be, for example, for the purpose of detecting altered viability, growth, or virulence resulting from the treatment.
  • Permissive location is meant a region of a protein molecule into which an additional peptide sequence (such as an aptamer sequence or a target molecule sequence) can be inserted, while allowing the protein molecule to retain sufficient biological function to allow cell survival.
  • Permissive location also refers to the corresponding region of a nucleic acid that encodes the above protein molecule, into which an oligonucleotide encoding the above peptide sequence may be inserted.
  • transformation is meant any method for introducing foreign molecules, such as DNA, into a cell (e.g., a bacterial, yeast, fungal, algal, plant, or animal cell).
  • a cell e.g., a bacterial, yeast, fungal, algal, plant, or animal cell.
  • Lipofection, DEAE-dextran-mediated transfection, microinjection, protoplast fusion, calcium phosphate precipitation, retro viral delivery, electroporation, natural transformation, and biolistic transformation are just a few of the methods known to those skilled in the art which may be -12- used.
  • Fig. 1 is a schematic diagram of the ABBIS system.
  • Fig. 2 is a diagram of the ABBIS- 1 vector and a diagram of a constrained 16mer combinatorial peptide within the disulfide loop of TrxA.
  • Fig. 3 is a diagram of plasmid pBMML2.
  • Fig. 4 is a diagram of the ABBIS-2 vector and a diagram of a constrained 16mer combinatorial peptide within a permissive loop of MalE.
  • Fig. 5 is a diagram of an arabinose disk diffusion assay for scoring toxic aptamers.
  • Figure 6 is a diagram showing growth curves for bacteria expressing the inhibitory aptamers KOI and KO52, compared to control bacteria not expressing these aptamers.
  • Fig. 7 is a diagram showing the arabinose-induced trimethoprin resistance and arabinose-induced thymine auxotrophy of E. coli clones containing ABBIS- 1 vectors that encode anti-ThyA aptamers.
  • ABS aptamer-based bacterial inhibition systems
  • Each ABBIS library is a combinatorial aptamer library composed of constrained random peptides.
  • the peptides are presented as loops of a scaffold protein, which serves to stabilize and display each random peptide within a cell.
  • Microbial cells such as those of bacteria, contain physically segregated subcellular compartments. Therefore, in order to access the full range of essential gene products that are potential drug target molecules, it is necessary to target the aptamers to these various subcellular compartments.
  • each aptamer library is generated using a specific targeting vector, which encodes a scaffold protein that intrinsically localizes to a specific subcellular compartment.
  • aptamers are expressed and localized to the appropriate subcellular compartment via scaffold protein localization.
  • Toxic or inhibitory aptamers bind to and inhibit the activity of target molecules that perform essential functions in, for example, the periplasm (Target 1) or the cytoplasm (Target 2) as shown in Fig. 1. Aptamer/target interactions are detected as decreases in cell growth, viability, or virulence.
  • ABBIS aptamer/target interaction
  • a toxic aptamer titrates the activity of its specific target molecule.
  • ABBIS is also useful as a means of producing aptamer-expressing cell strains for drug screening.
  • the activity of a given target molecule is only partially inhibited by an aptamer, resulting in the hypersensitization of the strain to other -14- drugs that inhibit the target molecule of interest.
  • the use of these hypersensitized screening strains increases the sensitivity of high-throughput screening assays for the identification and isolation of small molecule drugs that inhibit a novel or previously-known target molecule.
  • Target molecules may be protein, nucleic acid, lipid, or carbohydrate molecules that interact with a toxic aptamer in such a way that the natural function of the target is inhibited.
  • Target molecules once identified via ABBIS, can be used in conventional high-throughput screens for novel drugs that specifically bind and alter target molecule function in cell-free biochemical screens.
  • structural information i.e., obtained from nuclear magnetic resonance or X-ray crystallographical analysis
  • ABBIS X-ray crystallographical analysis
  • Aptamer-encoding synthetic gene sequences are produced as follows. DNA encoding random stretches of approximately five to thirty amino acids (e.g., combinatorial peptides) are generated by random oligonucleotide synthesis. The synthetic oligonucleotides are cloned into a permissive site within a gene encoding a scaffold protein (See Figs. 2 and 4). The cloning is performed in a way to ensure that the random oligonucleotide sequence is in frame with the scaffold protein coding sequence, resulting in expression of a scaffold protein/aptamer chimera. The scaffold protein- encoding gene is present (or subsequently placed) within an expression vector that is appropriate for the cell type in which the aptamer is to be expressed -15-
  • ABBIS Vector Design and Library Construction We have tested two types of ABBIS targeting vectors: one containing thioredoxin (TrxA) as a scaffold protein, and the other containing maltose binding protein (MalE) as a scaffold protein. These E. coli proteins intrinsically localize to the cytoplasmic and periplasmic compartments, respectively, of Gram-negative bacterial cells.
  • Our ABBIS targeting vectors have been engineered to allow insertion of short random peptides into either TrxA or MalE. Presumably, these inserted peptide "loops" are exposed at the surface of the folded chimeric scaffold protein, and are capable of interacting with target molecules.
  • any protein that tolerates insertion of random peptides may be used as a scaffold protein for construction of ABBIS libraries.
  • analogous aptamer targeting vectors that express scaffold proteins from Gram- positive bacterial cells, fungi (e.g., yeast), parasites, animal cells, plant cells, etc. may be used to target aptamers to subcellular compartments in these organisms in an analogous fashion.
  • An important goal of the ABBIS vector design is to ensure that aptamers are expressed only when it is desirable to do so, since toxic aptamer expression causes cell death or growth inhibition.
  • aptamer expression must be tightly regulated in order to fully exploit the ABBIS technology.
  • Chimeric aptamer-encoding genes cloned into the ABBIS vectors described below are under the tight transcriptional control of the araBAD promoter. Transcriptional activity of this promoter is strongly repressed in E. coli in the presence of glucose, and is strongly induced (more than 300-fold above basal levels) in the presence of 0.4% arabinose. Moreover, intermediate levels of araBAD promoter induction are obtained with intermediate arabinose concentrations (e.g., 0.001%). Unlike repressor-based transcription regulatory systems, regulation of the araBAD promoter is positive, i.e., transcriptional activation is dependent upon the presence of both the AraC regulatory protein and its activator, arabinose. Hence, spurious expression is less likely to occur than with repressor-dependent systems.
  • ABBIS vectors An additional useful feature of ABBIS vectors is the presence of a mobilization sequence derived from plasmid RK2.
  • the mobilization sequence allows highly efficient transfer of ABBIS libraries into various types of bacteria via conjugation.
  • any known technique for transformation of DNA into cells, whether natural or artificial, may be used.
  • ABBIS- 1 and ABBIS-2 vectors in ColEl based, and therefore, the vector is maintained at a high copy number in many Gram-negative bacterial species.
  • high copy vectors are not necessary, and vectors that are present within cells at low copy numbers also may be used for ABBIS.
  • analogous ABBIS vectors that are appropriate for use with other types of cells, such as bacterial, fungal, parasitic, animal, and plant cells, may be designed by selecting the appropriate plasmid replicons, regulatable expression systems, and scaffold proteins for subcellular targeting of aptamers; appropriate replicons, expression systems, scaffold proteins, and other necessary vector components are known to investigators who are skilled -17- in the art of molecular biology.
  • the ABBIS- 1 library was constructed in plasmid pBMT.
  • This library consists of plasmids that encode aptamers comprising 16 random amino acids (random 16mer peptides).
  • the aptamers are expressed within the context of the disulfide loop of E.coli thioredoxin (TrxA), and, hence, are targeted to the cytoplasm.
  • TrxA E.coli thioredoxin
  • the choice of 16 amino acids as the peptide length for construction of our initial ABBIS libraries was arbitrary: in practice, ABBIS vectors can accept inserts encoding virtually any small peptide.
  • pBMT the mobilization cassette from pBSL237 (Alexeyev and Shokolenko, Biotechniques 19: 22-26; 1995) was cloned into the Cla I site of pB AD 18 (Guzman et al., J. Bacteriol. Ill: 4121-4130; 1995) by ligation of fragments enzymatically rendered blunt-ended, and the resulting plasmid was designated pBADmob.
  • the trxA gene from E. coli strain XL- 1 Blue was cloned by PCR amplification between the EcoR I and Xba I sites of pBADmob.
  • oligonucleotides encoding random 16mer peptides were cloned into the unique Rsr II site (which begins after encoded amino acid 35 of Trx) of the trxA gene.
  • Fig. 2 shows a schematic diagram of a plasmid isolated from the ABBIS- 1 library, plasmid pBMTlOlQ. The amino acid sequence of the N- terminus of thioredoxin is shown, and the insertion of a random 16mer peptide after amino acid 35 of thioredoxin is denoted by a bracket.
  • the random 16mer peptide shown here comprises a portion of an aptamer that inhibits the function of thymidylate synthase.
  • the ABBIS-2 library was constructed in pBMML2 (Fig. 3).
  • This library consists of plasmids that encode random 16mer peptides expressed within the context of a permissive loop of the E. coli maltose binding protein (MalE), and, hence, aptamers are targeted to the periplasmic space.
  • the mobilization cassette from pBSL237 Alexeyev and Shokolenko, Biotechniques 19: 22-26; 1995
  • the resulting plasmid was designated pBADmob.
  • the malE allele containing a short deletion and insertion of a BamH I linker (malE133) from plasmid pPD178 (Martineau, et al., Gene 113: 35-46; 1992) was amplified by PCR, and the fragment was cloned into the Sma I site of pUC19.
  • the resulting plasmid was digested with BssH II and BstX I, and a synthetic oligonucleotide was inserted between these sites to reconstruct the original malE sequence, except that the BamH I site was replaced by adjacent Xho I and Xba I sites.
  • the altered malE gene was then excised and cloned between the EcoRl and Hind III sites of pBADmob (and is located between base pairs 1628 and 2850 of pBMML2).
  • a large stuffer fragment was cloned between the Xba I and Xho I sites to create plasmid pBMMLS.
  • the Xba I site inserted earlier into malE was replaced by a Bgl II site by replacing the DNA between the existing Bgl II and Xho I sites with a synthetic linker.
  • the Bgl II site within the original coding sequence o ⁇ malE was eliminated by replacing the 415 base pair fragment between the EcoR I and BstX I sites (base pairs 1628 and 2103 of pBMML2) with a PCR product in which the Bgl II site was eliminated (without altering the encoded amino acid sequence).
  • the resulting plasmid was designated pBMML2.
  • a large stuffer fragment was then cloned between the remaining Bgl II site and the adjacent Xho I site to create pBMML2S, which was used for -19- the construction of the ABBIS-2 library.
  • plasmid pBMML2 contains a malE gene whose sequence differs from the sequence of malEl 33 by a silent mutation of the Bgl II site, and by the replacement of the region around the BamH I site with the sequence shown below (the Bgl II and Xho I sites are underlined).
  • the "ala" codon shown below is replaced by a large stuffer, which is removed when the library is constructed.
  • the amino acid numbering corresponds to the numbering of the amino acids in the wild- type protein; amino acids without numbers are different from the wild-type sequence.
  • the stuffer region of pBMML2S was released by Bgl lllXho I digestion, and oligonucleotides encoding random 16mer peptides were cloned between the Bgl II and Xho I sites.
  • the random 16mer peptides were inserted into a region of the MalE protein (after amino acid 135) that tolerates large insertions without detrimental effect.
  • Fig. 4 shows a schematic diagram of a plasmid isolated from the ABBIS-2 library, plasmid pBMML2.12.
  • the bracketed amino acids comprise a portion of an aptamer that has no effect on the growth of the E. coli host, and, hence, serves as an ABBIS negative control.
  • the ABBIS- 1 library consists of 2.7 X 10 7 plasmids. DNA sequencing of the peptide-encoding region often randomly selected ABBIS- 1 plasmids showed that all ten plasmids encoded peptides of random sequence. Eight of the plasmids each contained an in- frame insertion consisting of a single oligonucleotide. The ninth plasmid contained an in-frame insertion consisting of two oligonucleotides encoding a 34 amino acid insertion ((2 X 16) + 2, because the sticky ends from the second oligonucleotide generate an additional two codons) that functions as an aptamer.
  • the tenth plasmid contained a single insertion that had a -1 frameshift mutation resulting in premature truncation of the scaffold protein/aptamer chimera.
  • Such truncated aptamers may also have activity (many of them appear to be toxic), but may be more difficult to work with on account of their small size.
  • DNA sequencing of two randomly selected plasmids from the ABBIS-2 library showed that these plasmids also encoded peptides of random sequence. Because 16 randomized amino acids can produce approximately 6 X 10 20 different peptides, the ABBIS- 1 and ABBIS-2 libraries represent only a small fraction of the total universe of random 16mer peptides. It is anticipated that more extensive libraries composed of 10 9 different aptamers are easily obtainable.
  • ABBIS vectors can accept inserts encoding any small peptide.
  • peptides within the size range of four to fifty amino acids in length will be the most useful, although peptides of other sizes also may be used.
  • Example I Isolation of toxic aptamers from the ABBIS- 1 library
  • the ABBIS- 1 library plasmid encodes thioredoxin fusion aptamers wherein each of the random peptides is inserted into the thioredoxin gene as an internal fusion after amino acid 35 of thioredoxin.
  • the position of each of these sequences within the protein is the same as the position of the random peptide in pBMTlOlQ (Fig. 2).
  • Toxic aptamers may be differentiated from non-toxic aptamers by comparing the growth of aptamer-encoding bacterial colonies on medium that induces aptamer expression (in this case, medium containing arabinose) versus control (non-inducing) medium.
  • the plate that contained bacteria expressing toxic aptamers displayed a clear zone (i.e., less bacterial growth) in the region of the agar into which arabinose had diffused, whereas no clear zone was observed for bacteria not expressing toxic aptamers.
  • This experiment confirms that arabinose-dependent growth inhibition occurs only when toxic aptamers are expressed.
  • the disc diffusion method allows the relative toxicity of an aptamer to be determined, since bacteria carrying aptamers of higher toxicity display larger zones of arabinose-induced growth inhibition.
  • the disc diffusion method also allows the selection of cells resistant to the effects of arabinose. We have observed colonies growing in the zone of inhibition near the arabinose disc.
  • Such cells may useful for the characterization of the mechanism of aptamer action. For example, if a given toxic aptamer kills cells by binding to a specific essential molecule (e.g., protein X), mutants that are resistant to the aptamer might contain mutations in protein X that block aptamer binding. These mutations can be mapped, cloned and identified by standard genetic approaches. We next screened about 4000 colonies for plasmids encoding toxic aptamers by replica plating the colonies from medium lacking arabinose to medium containing 0.4% arabinose.
  • a specific essential molecule e.g., protein X
  • Two candidates were identified, each of which had a single insert, one of which was in frame, and one of which contained a -1 frameshift mutation.
  • Two of the clones contained an in- frame, double oligonucleotide insert, 31 of the clones contained a -1 frameshift mutation within the insert, and one of the clones contained a -2 frameshift mutation within the insert.
  • JB63 IRMCLRWSCFCLLTLV (SEQ ID NO: 3) KOI LWFTEVRGHGWRYKVG (SEQ ID NO: 4)
  • Figure 6 shows growth curves for bacteria expressing the inhibitory aptamers KOI and KO52 shown above, compared to control bacteria not expressing these aptamers.
  • Overnight cultures of E. coli SM10 carrying a plasmid encoding either KOI or KO52 were diluted 10,000-fold into Luria-Bertani medium containing 100 ⁇ g/ml ampicillin. The cultures were shaken at 37° C for 2 hours, after which arabinose was added to a final -24- concentration of 0.1%. Control cultures did not receive arabinose. Samples from experimental and control cultures were removed at the times indicated in Fig. 6 and titers were determined on Luria-Bertani agar plates containing 100 ⁇ g/ml ampicillin.
  • arabinose-induced expression of aptamer KOI appears to be bacteriocidal
  • arabinose-induced expression of aptamer KO52 appears to be bacteriostatic
  • arabinose-induced expression of aptamer trxAl 19 which does not inhibit bacterial growth on solid medium, similarly did not inhibit growth in liquid culture.
  • Thymidylate synthase is an essential enzyme in the pathway for biosynthesis of deoxythymidine monophosphate (dTMP) from deoxyuridine monophosphate (dUMP). Mutants defective in Thy A cannot grow on minimal medium that lacks exogenously added thymine; however, the addition of 0.01% thymine to minimal medium allows the growth of Thy A mutants. Mutants deficient in ThyA are resistant to the antibiotic trimethoprim when grown on medium containing thymine. Thus, growth on minimal medium with trimethoprim and thymine provides a positive selection for the ThyA-deficient phenotype.
  • E. coli SM10 containing the ABBIS- 1 library on minimal medium containing 0.01% thymine, 1 ⁇ g/ml trimethoprim, and 0.01% arabinose, and incubated it for 48 hours at 37° C.
  • Fig. 7 shows an example of an experiment in which anti-ThyA -26- aptamers were tested for their ability to confer arabinose-induced trimethoprin resistance and thymine auxotrophy.
  • E. coli ThyA + strain SM10 carrying ABBIS- 1 plasmids encoding either weakly or strongly inhibitory anti-ThyA aptamers, and control ThyA + and ThyA "
  • coli SM10 strains carrying plasmids that encode inactive aptamers were uniformly streaked out onto defined medium containing 50 ⁇ g/ml ampicillin and various combinations of 0.01% arabinose, 10 ⁇ g/ml thymine and 1 ⁇ g/ml trimethoprim, as indicated in Fig. 7. Plates were incubated at 37° C for approximately 35 hours.
  • Fig. 7 shows that anti-ThyA aptamers confer trimethoprim resistance only in the presence of arabinose. Moreover, when arabinose is present and thymine is absent, the strain expressing the weakly inhibitory aptamer grows poorly, and the strain expressing the strongly inhibitory aptamer does not grow at all (the few colonies seen in the strong aptamer sample were confirmed to contain mutant plasmids that had lost their inhibitory properties).
  • the aptamer-dependent growth inhibition observed on minimal plates containing arabinose could be completely suppressed by the presence of thymine, confirming that the aptamer-dependent growth inhibition was ThyA-specific (the Thy + control strain grows well under the "Minimal + Arabinose” and “Minimal + Arabinose + Thy” conditions, but its growth is less apparent in the bottom two panels of Fig. 7, because the Thy + control strain was less-thickly streaked than the other strains).
  • Fig. 2 shows the deduced peptide sequence of the strongly inhibitory anti-ThyA aptamer (SYLCVTPASQLTEFWG; SEQ ID NO: 1).
  • the DNA sequence encoding this peptide was cloned into the ABBIS- 1 vector.
  • This plasmid (pBMTlOlQ) was transformed into various strains of E. coli, and its ability to confer arabinose-dependent trimethoprim resistance and thymine auxotrophy was confirmed.
  • pBMTlOlQ This plasmid
  • ABBIS technology also may be used to inhibit eukaryotic enzymes for which there are microbial homologues.
  • a screening strain of E. coli whose growth depends on the human ThyA product, by replacing the E. coli thyA gene with the human thyA homologue.
  • Such a strain could then be used in an ABBIS selection to find aptamers that inhibit the human ThyA protein.
  • the aptamers could be used to generate hypersensitive screening strains for high throughput drug screening (see below) or as a starting point for structure-based, molecular drug design.
  • the aptamers themselves (or modified versions thereof) may be useful pharmaceutical compounds. Drugs discovered using these approaches should be useful for the treatment of disease involving cell proliferation, such as cancer, autoimmune disease, and psoriasis.
  • the ThyA protein can be converted to a substrate for HIV protease by insertion of a "loop" of HIV substrate peptide into a permissive location within the ThyA protein (ThyA-HIVS). Expression of the HIV protease in bacterial cells whose sole source of ThyA is chimeric ThyA-HIVS results in the ThyA " phenotype (because the protease cleaves ThyA-HIVS). Thus, the
  • ABBIS system may be used to discover aptamers that inhibit HIV protease (and by analogy, any other protease) by selecting for the arabinose-dependent, ThyA + phenotype after introducing the ABBIS plasmid library into the strain expressing both the HIV protease and the ThyA-HIVS (or another protease and artificial protease substrate).
  • ABBIS may be used to discover aptamer inhibitors of any target protein regardless of its origin (e.g., -28- mammalian, viral, parasitic) by engineering bacterial strains in which the function of the target protein can be easily scored or modified by a selectable phenotype.
  • Such inhibitory aptamers can then be used as described above as probes for structure-based, molecular drug design.
  • ABBIS may be used in gene therapy protocols to inhibit the function of target proteins that cause or are involved in disease processes.
  • Aptamer expression vectors may be engineered for optimal regulation of aptamer expression and subcellular targeting by methods that are known to those who are skilled in the arts of molecular biology, genetic engineering, and gene therapy.
  • Example III Construction of an antibiotic hypersensitive screening strain that expresses toxic aptamers at subinhibitory levels
  • the target molecules of toxic aptamers identified in our preliminary screens of ABBIS libraries are currently unknown.
  • some of these toxic aptamers might inhibit cell growth by interfering with membrane functions (such as transport) or cell envelope integrity, by inhibiting essential target proteins involved in these cell processes and properties.
  • a "permeabilizing" aptamer might render cells, such as bacteria and fungi, hypersensitive to antibiotics and other antimicrobial agents. Such aptamers would be useful for constructing screening strains that would facilitate the identification of new antimicrobial drugs.
  • coli that encode inhibitory Aptamer 7 display a higher sensitivity to gentamycin when grown in the presence of subinhibitory concentrations of arabinose than when grown in the absence of arabinose; the increased sensitivity results in a relatively larger zone of growth inhibition surrounding gentamycin-impregnated disks.
  • toxic aptamers will be useful for constructing drug-hypersensitive screening strains that will enhance the sensitivity of high-throughput drug screens, compared to those screens that employ unmodified bacterial strains.
  • Such hypersensitive strains will be useful for detecting low concentrations of bioactive drugs that are present within complex mixtures.
  • Screening strains may be constructed using aptamers that inhibit unknown target molecules (such as Aptamer 7), or known target molecules.
  • a screening strain may be constructed using plasmid pBMTlOlQ (described above), which encodes a strongly inhibitory anti-ThyA aptamer.
  • the appropriate arabinose concentration that induces sub-inhibitory expression of the anti-ThyA aptamer can be readily determined.
  • the strain, when grown under hypersensitizing conditions e.g., those that induce sub-inhibitory aptamer expression
  • -30- Table I Effects of aptamer expression on growth inhibition by gentamicin
  • E.coli E.coli
  • strain SM10 E.coli
  • plasmids that encode the indicated aptamers were spread onto LB agar with ampicillin, with or without 0.005% arabinose (0.005% arabinose induces sub-maximal levels of aptamer expression).
  • a disc impregnated with 10 ⁇ g of gentamicin was placed on the plate, which was then incubated overnight. The zone of inhibition around the gentamicin disc was measured, and the diameter is shown above. The larger the zone, the lower the concentration of gentamicin required to inhibit growth.
  • the control plasmid encodes an aptamer that is not toxic to the cells.
  • Example IV Selection for aptamers that inhibit the function of virulence factors, improvement of aptamers and conversion to drugs, and identification of aptamer targets by genetic and biochemical approaches
  • toxic ABBIS aptamers may be identified by screening for general bacterial growth inhibition, or by screening for inhibition of a specific essential target molecule such as ThyA.
  • useful ABBIS aptamers may be identified using target molecules that are not essential for bacterial growth and survival, or that are essential only under certain conditions. Such targets may, however, play important roles in infection and disease.
  • bacterial virulence factors constitute a group of proteins (e.g., toxins, enzymes, or regulatory proteins) or structures (e.g., pili, flagella, capsules, or secretion systems) that are not essential for bacterial viability on artificial media.
  • proteins e.g., toxins, enzymes, or regulatory proteins
  • structures e.g., pili, flagella, capsules, or secretion systems
  • virulence factors frequently are essential for growth and survival in host tissues.
  • An antimicrobial compound that interferes with the biological function of a virulence factor would have clinical value in the treatment of infection.
  • ABBIS may be used for the isolation of aptamers that inhibit nonessential or conditionally essential target molecules, such as bacterial virulence factors, by screening for arabinose- dependent loss of virulence.
  • In vitro and in vivo screens for virulence factor function are known to those of skill in the art of microbiology.
  • the desired activity of a given aptamer may be improved by performing repeated cycles of random mutagenesis of an aptamer coding sequence followed by selection of the desired activity under decreasing arabinose concentrations.
  • This mutational modification and selection of aptamers also provides a means by which to define critical residues that are -32- responsible for aptamer activity.
  • peptide derivatives that contain the minimal inhibitory sequence plus additional chemical moieties may be synthesized.
  • Such functional groups e.g., cyclic bonds, peptide bond mimics, etc.
  • Such moieties can enhance the toxic activity of an aptamer, thus increasing its usefulness as an antimicrobial compound.
  • host bacteria can be chemically mutagenized prior to introduction of a plasmid encoding a known aptamer. Mutants that resist the action of a given aptamer can be selected, and the gene that confers resistance against an aptamer can be cloned and mapped by standard genetic complementation methods. DNA sequencing can be used to identify the mutations that allowed the target molecule to escape from the aptamer' s inhibitory activity, assuming that resistance to an aptamer results from mutations in the aptamer target molecule itself.
  • Biochemical binding of an aptamer to its target molecule may be assessed by passing cell extracts over an aptamer-derivatized column.
  • radiolabelled or enzyme-labelled aptamers could be used as probes to isolate target molecules (for example, using a protocol analagous to a Western blot). Extracts of mutant, aptamer-resistant cells would be useful controls in such experiments.
  • standard drug discovery methods and high throughput screening can -33- be used to find small molecule drugs that inhibit the target molecule.
  • sequences of a combinatorial peptide that are crucial for the inhibitory activity of a given aptamer can be determined by construction of substitution or deletion mutations, followed by structure-function analyses, and analyses of aptamer/target interactions. Ultimately, crystallization of complexes between an aptamer-derived peptide and its target molecule will provide the most detail for each aptamer/target interaction. Such information would accelerate the synthesis of aptamer analogs (i.e., peptide mimetics) that are useful as antimicrobial drugs.
  • Example V Selection for aptamers that inhibit or stimulate the PhoBR signal transduction pathway
  • PhoBR The PhoBR signal transduction pathway regulates phosphate concentration-dependent gene expression (for review, see B. L. Wanner, in: Metal Ions in Gene Regulation, S. Silver and W. Walden, eds., Chapman and Hall, 1997, pp. 104-128).
  • PhoBR is a two-component pathway requiring a phosphate sensor molecule, PhoR, and a transcriptional activator molecule, PhoB.
  • PhoR a transmembrane histidine kinase
  • PhoB a transmembrane histidine kinase
  • Phosphorylation of PhoB leads to transcriptional activation of at least eleven phosphate-regulated genes.
  • PhoR also possesses a phosphatase activity that deactivates phospho-PhoB when extracellular phosphate increases. This negative regulation is dependent on PstSCAB, a phosphate-specific transporter, and PhoU, a peripheral membrane protein.
  • these reagents may be used in high-throughput assays to isolate compounds that mimic a given apatamer's ability to bind a given essential target molecule.
  • Compounds identified by such approaches may have utility as therapeutic agents in inhibiting the viability, growth, or virulence of undesired -36- cell or microorganisms.
  • samples containing an essential target molecule of interest are loaded onto the wells of microtiter plates coated with an aptamer known to bind to and inhibit the biological activity of the essential target molecule. Unbound antigen is washed out, and an antibody that specifically recognizes the essential target molecule, coupled to an agent to allow for detection, is added.
  • Agents allowing detection include alkaline phosphatase (which can be detected following addition of colorimetric substrates such as - nitrophenolphosphate), horseradish peroxidase (which can be detected by chemiluminescent substrates such as ECL, commercially available from Amersham) or fluorescent compounds, such as FITC (which can be detected by fluorescence polarization or time-resolved fluorescence).
  • the amount of antibody binding, and hence the relative amount of aptamer bound to the essential target molecule in a sample, is easily quantitated on a microtiter plate reader. It is understood that appropriate controls for each assay are always included as a baseline reference. A decrease in the amount of aptamer/essential target molecule binding suggests that a test compound may bind to the essential -37- target molecule.
  • a positive assay result that is, identification of a compound that mimics the binding of a inhibitory aptamer to an essential target molecule, is indicated by a decrease of at least 2-fold (preferably, at least 3-fold, more preferably, at least 5-fold, still more preferably, at least 7-fold, and most preferably, at least 10-fold) in the level of essential target molecule/aptamer complexes.
  • test compound disrupts an essential target molecule/aptamer interaction as of a result of its binding to the essential target molecule, rather than to the aptamer, the test compound may be bound to a solid support, and its binding affinity for the essential target molecule versus the aptamer may be determined.
  • a test compound that disrupts the essential target molecule/aptamer interaction by binding the essential target molecule will have a higher affinity for the essential target molecule than for the aptamer.
  • High-throughput assays for the purpose of identifying compounds that mimic the binding activity of a given aptamer isolated by the methods of the invention can be performed using aptamers and/or essential target molecules within cell lysates, or as purified or partially-purified molecules. It is understood that variations of the assay described above, or other binding assays that will be apparent to those of ordinary skill in the art, may be employed to identify compounds that mimic the binding activity of an inhibitory aptamer to its cognate essential target molecule. For, example, in one variation of the ELISA assay described above, the microtiter wells are coated with the essential target molecule and the level of essential target molecule/aptamer interaction is detected by using an antibody that specifically binds the aptamer. -38-
  • Compounds that are identified as having the ability to bind essential target molecules may be tested for their ability to inhibit the biological activity (e.g., enzymatic activity) of such molecules or to inhibit the growth, viability, or virulence of a cell or microorganism by methods that are known to those of ordinary skill in the art.
  • biological activity e.g., enzymatic activity
  • novel drugs that bind to and inhibit the biological activity of essential target molecules or inhibit the viability, growth, or virulence of an organism are identified from large libraries of both natural products or synthetic (or semi-synthetic) extracts or chemical libraries according to methods known in the art.
  • test extracts or compounds are not critical to the screening procedure(s) of the invention. Accordingly, virtually any number of chemical extracts or compounds can be screened using the exemplary methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds.
  • Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, NH) and Aldrich Chemical (Milwaukee, WI).
  • libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch -39-
  • the goal of the extraction, fractionation, and purification process is the careful characterization and identification of a chemical entity within the crude extract having the ability to bind to and inhibit the biological activity of at least one essential target molecule or having the ability to inhibit the viability, growth, or virulence of an organism.
  • the same assays described herein for the detection of activities in mixtures of compounds can be used to purify the active component and to test derivatives thereof. Methods of fractionation and purification of such heterogenous extracts are known in the art.
  • compounds shown to be useful agents for treatment are chemically modified according to methods known in the art. Compounds identified as being of therapeutic value may be subsequently analyzed, if appropriate, using one of many animal models known to be useful for -40- pharmaceutical testing.
  • the compounds (inhibitory aptamers and small molecule compounds that mimic the binding properties of inhibitory aptamers) identified using any of the methods disclosed herein, may be administered to patients or experimental animals with a pharmaceutically-acceptable diluent, carrier, or excipient, in unit dosage form.
  • a pharmaceutically-acceptable diluent, carrier, or excipient Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer such compositions to patients or experimental animals.
  • intravenous administration is preferred, any appropriate route of administration may be employed, for example, parenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, or oral administration.
  • Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops
  • Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes.
  • Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds.
  • Other potentially useful parenteral delivery systems for antagonists or agonists of the invention include ethylene- vinyl acetate copolymer particles, osmotic pumps, -41- implantable infusion systems, and liposomes.
  • Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.

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Abstract

L'invention a trait à des procédés d'identification d'aptamères peptidiques qui se lient à des molécules cibles essentielles et inhibent la viabilité, la croissance ou la virulence d'un organisme, à des procédés d'isolement de composés qui imitent l'activité de ces aptamères peptidiques, et à des aptamères peptidiques identifiés par les procédés de l'invention.
PCT/US1999/006466 1998-03-27 1999-03-26 Systemes d'inhibition bacteriens a base d'aptameres (abbis) WO1999050462A1 (fr)

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WO2002006517A2 (fr) * 2000-07-19 2002-01-24 Kairos Scientific, Inc. Methodes a haut rendement permettant de preparer et de cribler des composes qui affectent la viabilite cellulaire
WO2003060142A2 (fr) * 2001-10-18 2003-07-24 Essentia Biosystems, Inc. Compositions et procede pour liberation controlee
US7300922B2 (en) 2001-05-25 2007-11-27 Duke University Modulators of pharmacological agents
US7304041B2 (en) 2004-04-22 2007-12-04 Regado Biosciences, Inc. Modulators of coagulation factors
US7312325B2 (en) 2000-09-26 2007-12-25 Duke University RNA aptamers and methods for identifying the same

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US5270170A (en) * 1991-10-16 1993-12-14 Affymax Technologies N.V. Peptide library and screening method
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Cited By (20)

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US6924101B2 (en) 1997-11-14 2005-08-02 San Diego State University Foundation Methods for identifying anti-microbial agents
US6228579B1 (en) 1997-11-14 2001-05-08 San Diego State University Foundation Method for identifying microbial proliferation genes
WO2002006517A2 (fr) * 2000-07-19 2002-01-24 Kairos Scientific, Inc. Methodes a haut rendement permettant de preparer et de cribler des composes qui affectent la viabilite cellulaire
WO2002006517A3 (fr) * 2000-07-19 2002-09-19 Kairos Scient Inc Methodes a haut rendement permettant de preparer et de cribler des composes qui affectent la viabilite cellulaire
US7741307B2 (en) 2000-09-26 2010-06-22 Duke University RNA aptamers and methods for identifying the same
US7776837B2 (en) 2000-09-26 2010-08-17 Duke University RNA aptamers and methods for identifying the same
US8143233B2 (en) 2000-09-26 2012-03-27 Duke University RNA aptamers and methods for identifying the same
US7858591B2 (en) 2000-09-26 2010-12-28 Duke University RNA aptamers and methods for identifying the same
US7312325B2 (en) 2000-09-26 2007-12-25 Duke University RNA aptamers and methods for identifying the same
US7812001B2 (en) 2000-09-26 2010-10-12 Duke University RNA aptamers and methods for identifying the same
US7776836B2 (en) 2000-09-26 2010-08-17 Duke University RNA aptamers and methods for identifying the same
US7300922B2 (en) 2001-05-25 2007-11-27 Duke University Modulators of pharmacological agents
US8283330B2 (en) 2001-05-25 2012-10-09 Duke University Modulators of pharmacological agents
US8586524B2 (en) 2001-05-25 2013-11-19 Duke University Modulators of pharmacological agents
WO2003060142A3 (fr) * 2001-10-18 2004-02-12 Essentia Biosystems Inc Compositions et procede pour liberation controlee
WO2003060142A2 (fr) * 2001-10-18 2003-07-24 Essentia Biosystems, Inc. Compositions et procede pour liberation controlee
US7723315B2 (en) 2004-04-22 2010-05-25 Regado Biosciences, Inc. Modulators of coagulation factors
US7304041B2 (en) 2004-04-22 2007-12-04 Regado Biosciences, Inc. Modulators of coagulation factors
US8389489B2 (en) 2004-04-22 2013-03-05 Regado Biosciences, Inc. Modulators of coagulation factors
US8859518B2 (en) 2004-04-22 2014-10-14 Regado Biosciences, Inc. Modulators of coagulation factors

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