WO2006108225A1 - Procede de criblage pour des composes qui modulent la proliferation cellulaire - Google Patents

Procede de criblage pour des composes qui modulent la proliferation cellulaire Download PDF

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WO2006108225A1
WO2006108225A1 PCT/AU2006/000489 AU2006000489W WO2006108225A1 WO 2006108225 A1 WO2006108225 A1 WO 2006108225A1 AU 2006000489 W AU2006000489 W AU 2006000489W WO 2006108225 A1 WO2006108225 A1 WO 2006108225A1
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cortactin
compound
expression
candidate compound
protein
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Roger Daly
Ashleigh Wilson
Paul Timpson
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Garvan Institute Of Medical Research
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to a method of screening for compounds that modulate cell proliferation, and to compounds identified by these methods.
  • the invention further relates to reducing or enhancing cell proliferation in subjects in need thereof. Also encompassed by the present invention are methods for prognostic and diagnostic evaluation of diseases associated with aberrant cell proliferation.
  • Cancer is a heterogeneous group of diseases presenting in various forms in various tissues but having in common the characteristic of uncontrolled cell proliferation. Indeed, for some time, cancer has been recognized as a disease of uncontrolled cell proliferation. Thus, the rapidly proliferating cell has been the target of cancer chemotherapy. The goal is to find agents that are more effective against rapidly proliferating cells than against normal cells.
  • Cortactin is a multi-domain actin-binding protein that is probably tyrosine phosphorylated by a Src kinase, that has been shown to bind several other proteins, including proteins of the Arp2/3 complex (see below), brain cortactin binding protein- 1 (CortBPl or Shank 2; Du et ⁇ /., MoI. Cell. Biol. 18, 5838-5851, 1998; Weed and Parsons Oncogene 20, 6418-6434, 2001), brain ZO-I protein (Katsube et aL, J. Biol. Chem 273, 29672-29677, 1998), and dynamin-2 (McNiven et al, J. Cell Biol.
  • cortactin contributes to the spatial organization of sites of actin polymerization, coupled to selected cell surface transmembrane receptor complexes. Accordingly, cortactin is thought to be involved in one or more signalling pathways that regulate cell-cell contacts and the motogenic properties of cells.
  • Cortactin is normally enriched within the lamellipodia of motile cells and in neuronal growth cones, particularly in the brain. Cortactin is localized with the actin-related protein (Arp) 2/3 complex, at sites within the lamellipodia where actin polymerizes. Cortactin stimulates nucleation activity of the Arp2/3 complex, and enhances actin polymerization induced by proteins associated with Wiskott-Aldrich Syndrome (WASPs), that are co-activators of the Arp2/3 complex (Weaver et at. Curr. Biol. 11, 37-374, 2001; Urono et al, Nature Cell Biol. 3, 259-266, 2001).
  • WASPs Wiskott-Aldrich Syndrome
  • cortactin promotes the formation and stability of branched actin networks.
  • cortactin may regulate vesicle trafficking, for example, of endosomes (Kaksonen et al, J. Cell Sci. 113, 4421-4426, 2000), a role that is supported by its interaction with dynamin-2 (McNiven et al, J. Cell Biol. 151, 187-198, 2000).
  • cortactin also appears to regulate the organization and subcellular localization of transmembrane complexes, wherein CortBPl performs a scaffolding function in the organization of receptor complexes at post-synaptic sites of excitatory synapses, and ZO-I interacts with the transmembrane proteins claudin and occludin at epithelial tight junctions (Weed and Parsons Oncogene 20, 6418-6434, 2001). Cortactin also binds to rpde6, in rat brain tissue. " ' " " "• "'
  • the amino acid sequence of the cortactin polypeptide comprises the following domains: (i) an N-terminal stretch of acidic amino acid residues (NTA); (ii) Six to seven tandem repeats of an amino acid sequence that is related to that found in HSl, located C-terminal to the acidic domain; (iii) a sequence having the predicted structure of an ⁇ -helix located C-terminal to the HsI /cortactin repeat; (iv) a region rich in serine, threonine and proline, located C-terminal to the ⁇ -helical domain; and (v) a C-terminal SH3 domain.
  • cortactin Over expression of cortactin also enhances cell motility and invasion in vitro (Patel et al, Oncogene 16, 3227-3232, 1998; Huang et al, J. Biol. Chem. 273, 25770-25776, 1998). Over expression of cortactin in breast cancer cells may also affect the invasive properties of the cancer cells (Kairouz and Daly, Breast Cancer Res 2, 197-202, 2000), and metastases in bone (Li et al, Cancer Res. 61, 6906-6911, 2001).
  • the EMSl gene that encodes cortactin is commonly amplified in breast cancers and squamous cell carcinomas of the head and neck (Schuuring et al, Cancer Res.
  • cortactin has now found that reduction of cortactin expression in cancer cell lines inhibits cell proliferation. This finding indicates for the first time that cortactin plays a role in cell division or proliferation, and that agents that modulate the activity or expression of cortactin are potential therapeutics for diseases associated with aberrant cell proliferation. In other words, cortactin has now been identified as a target to use in screening for therapeutic agents that modulate cell division or proliferation.
  • the present invention provides a method of screening for an agent that modulates cell proliferation, the method comprising analysing the activity or expression of cortactin in the presence of a candidate compound, wherein altered activity or expression of cortactin in the presence of the compound when compared to the absence of the compound indicates that the compound is a potential modulator of cell proliferation.
  • the present invention also provides a candidate compound identified by a screening method of the present invention.
  • the present invention also provides a method for modulating cell proliferation, the method comprising administering to the cell population a compound that modulates the activity or expression of cortactin in an amount effective to modulate cell proliferation.
  • the present invention also provides a composition for inhibiting cell proliferation, the composition comprising an antiproliferative agent selected from the group consisting of a peptide derived from cortactin, a cortactin dominant-negative mutant, an antibody directed against cortactin, antisense compounds directed against cortactin-encoding mRNA, anti-cortactin catalytic molecules such as ribozymes or a DNAzymes and dsRNA or RNAi molecules that target cortactin expression or combinations of any of these agents.
  • an antiproliferative agent selected from the group consisting of a peptide derived from cortactin, a cortactin dominant-negative mutant, an antibody directed against cortactin, antisense compounds directed against cortactin-encoding mRNA, anti-cortactin catalytic molecules such as ribozymes or a DNAzymes and dsRNA or RNAi molecules that target cortactin expression or combinations of any of these agents.
  • the present invention also relates to methods for detecting the expression of cortactin polynucleotides or polypeptides in a sample (e.g., tissue or sample). Such methods can, for example, be utilized as part of prognostic and diagnostic evaluation of diseases associated with aberrant cell proliferation and for the identification of subjects exhibiting predispositions to such disorders.
  • the present invention provides a method of testing for a condition associated with aberrant cell proliferation in a human or animal subject, the method comprising contacting a biological sample from the subject being tested with an agent which binds to a cortactin polynucleotide or polypeptide for a time and under conditions sufficient for binding to occur and then detecting the binding wherein a modified level of binding of the agent for the subject being tested compared to the binding obtained for a control subject not having a condition associated with aberrant cell proliferation indicates that the subject being tested has a condition associated with aberrant cell proliferation.
  • the present invention also provides a method of monitoring the efficacy of a therapeutic treatment of a disease associated with aberrant cell proliferation, the method comprising:
  • kits comprising polynucleotide probes and/or monoclonal antibodies, and optionally quantitative standards, for carrying out methods of the invention. Furthermore, the invention provides methods for evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of disorders as recited herein.
  • FIG. 1 Alteration ofcortactin expression in head and neck squamous cell carcinoma cell lines:
  • A Western blot analysis of cortactin expression in a panel of HNSCC cell lines.
  • Scc9 cells exhibit a normal EMSl gene copy number, while FaDu and Detroit cells are I lql3-amplified.
  • Sccl5 cells overexpress cortactin in the absence of EMSl gene amplification;
  • B Suppression of cortactin expression in FaDu cells by transfection with a pSiren construct encoding a cortactin-selective shRNA.
  • FIG. 2A Cortactin overexpression enhances cell proliferation in HNSCC cells. FaDu ( Figure 2A) or Detroit 562 ( Figure 2B) cell ' s transfected with cortactin or lamin siRNA, and control or cortactin-overexpressing Scc9 cells ( Figure 2C) were subjected to cell proliferation assays in 10% FCS/EMEM. The cell number was estimated using a CellTiter 96 ® Aqueous Non-radioactive Cell Proliferation assay (Promega, Madison, WI) following the manufacturer's protocol and is expressed in arbitrary units (MTT units). The data points are the mean of 6 replicates.
  • FIG. 3 Inhibition ofcortactin expression reduces cell proliferation in HNSCC cells.
  • Detroit 562 cells were transfected with cortactin or lamin siRNA and then grown in Eagle's minimal essential medium (EMEM) supplemented with fetal calf serum (FCS) at a concentration of 0.25% (A) 5 1% (B) and 2.5% (C) for 5-6 days.
  • EMEM Eagle's minimal essential medium
  • FCS fetal calf serum
  • the cell number was estimated using a CellTiter 96 ® Aqueous Non-radioactive Cell Proliferation assay (Promega, Madison, WI) following the manufacturer's protocol and is expressed in arbitrary units (MTT units). The data points are the mean of 6 replicates.
  • Figure 4 Effect of suppression of cortactin expression on cell proliferation.
  • Detroit 562 (A) or FaDu cells (B), growing in 10% FCS were transfected with control (Lamin A/C) or cortactin-specific siRNAs. DNA content of the cells was then determined 3 days later by flow cytometry and the percentage of cells in S -phase expressed relative to that in control cells. * p ⁇ 0.05, indicating a significant difference between control and cortactin siRNA-treated cells.
  • FIG. 5 Effect of suppression of cortactin expression on the expression and activation of specific signalling proteins.
  • Cell lysates prepared from cells treated as described in Figure 4 were subject to Western blot analysis with the indicated antibodies.
  • Cortactin promotes colony formation by HNSCC cells.
  • Two cell lines expressing high levels of cortactin, Detroit 562 (A) and FaDu (B) were treated with control or cortactin-selective siRNAs.
  • two cell lines expressing low levels of cortactin, Scc25 (C) and Scc9 (D) were transfected with an empty vector or a cortactin expression vector. The cells were then plated in 6 well plates and fixed and stained 14 days later (Scc25 and Scc9) or upon reaching confluence (Detroit 562 and FaDu).
  • Cortactin expression levels Expression of cortactin in NIH3T3 pools infected with the vector control retrovirus (V) or a cortactin-encoding retrovirus (C).
  • B Focus formation assays. The cell pools described in A were subject to a focus assay and then fixed and stained.
  • SEQ ID NO: 3 mouse cortactin.
  • SEQ ID NO: 4 - cDNA encoding mouse cortactin.
  • SEQ ID NO's 5 to 7 Polynucleotides for producing siRNA molecules which downregulate human cortactin production (as described in Example 1).
  • SEQ ID NO: 9 to 29 Polynucleotides for producing siRNA molecules which target human cortactin as predicted by siRNA prediction software (siRNA Target Finder; http ://www.ambion. com/techlib/misc/siRNA finder.htmD .
  • the present invention provides a method of screening for an agent that modulates cell proliferation, the method comprising analysing the activity or expression of cortactin in the presence of a candidate compound, wherein altered activity or expression of cortactin in the presence of the compound when compared to the absence of the compound indicates that the compound is a potential modulator of cell proliferation.
  • modulation of cell proliferation refers to any change in the proliferation of a cell, when compared to a control cell of the same type. For example, this term can be used to describe an increase or a decrease in the rate of cell division.
  • a modulation of proliferation may refer to a normally quiescent cell entering into the cell cycle or a normally dividing cell ceasing to enter into the cell cycle.
  • reduced cortactin activity or expression in the presence of the compound indicates that the compound is a potential antiproliferative agent.
  • antiproliferative agent we mean an agent that reduces or inhibits cell proliferation.
  • the present invention provides a method of screening for an antiproliferative agent, the method comprising analysing the activity or expression of cortactin in the presence of a candidate compound, wherein reduced activity or expression of cortactin in the presence of the compound when compared to the absence of the compound indicates that the compound is an antiproliferative agent.
  • enhanced cortactin activity or expression in the presence of the compound indicates that the compound is an agent that enhances cell proliferation.
  • the present invention provides a method of screening for an agent that enhances cell proliferation, the method comprising analysing the activity or expression of cortactin in the presence of a candidate compound, wherein increased activity or expression of cortactin in the presence of the compound when compared to the absence of the compound indicates that the compound is an agent that enhances cell proliferation.
  • the method involves analysing the expression of cortactin in the presence of a candidate compound.
  • the amino acid sequences of the human and mouse cortactin polypeptides are exemplified herein as SEQ ID Nos: 1 and 3, respectively.
  • the term "cortactin” shall be taken to mean any peptide, polypeptide, or protein having at least about 80% amino acid sequence identity to the amino acid sequence of the human or mouse cortactin polypeptide set forth in SEQ ID NO: 1 or 3.
  • the percentage identity to SEQ ID NO: 1 or 3 is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%.
  • the cortactin is human cortactin.
  • cortactin shall also be taken to include a peptide, polypeptide or protein having the known biological activity of cortactin, or the known binding specificity of cortactin, wherein said peptide, protein or polypeptide is further capable of binding to a known binding partner of cortactin as described herein.
  • amino acid identities and similarities are calculated using software of the Computer Genetics Group, Inc., University Research Park, Maddison, Wisconsin, United States of America, e.g., using the GAP program of Devereaux et al, Nncl Acids Res. 12, 387-395, 1984, which utilizes the algorithm of Needleman and Wunsch, J MoI. Biol. 48, 443-453, 1970.
  • the CLUSTAL W algorithm of Thompson et al, Nucl. Acids Res. 22, 4673-4680, 1994 is used to obtain an alignment of multiple sequences, wherein it is necessary or desirable to maximize the number of identical/similar residues and to minimize the number and/or length of sequence gaps in the alignment.
  • BLAST Basic Local Alignment Search Tool
  • NCBI National Center for Biotechnology Information
  • BLAST 2 Sequences a tool that is used for direct pairwise comparison of two nucleotide sequences.
  • NCBI Network Codebook
  • nucleotide sequence falls within a particular percentage identity limitation recited herein, those skilled in the art will be aware that it is necessary to conduct a side-by-side comparison or multiple alignment of sequences. In such comparisons or alignments, differences may arise in the positioning of non- identical residues, depending upon the algorithm used to perform the alignment.
  • reference to a percentage identity between two or more nucleotide sequences shall be taken to refer to the number of identical residues between said sequences as determined using any standard algorithm known to those skilled in the art.
  • nucleotide sequences may be aligned and their identity calculated using the BESTFIT program or other appropriate program of the Computer Genetics Group, Inc., University Research Park, Madison, Wisconsin, United States of America (Devereaux et al, Nucl. Acids Res. 12, 387-395, 1984).
  • BLAST is also useful for aligning nucleotide sequences and determining percentage identity.
  • analysing the expression of cortactin involves determining the amount of cortactin protein and/or a cortactin-associated polynucleotide transcript. In one preferred embodiment, the amount of cortactin protein is measured using an anti- cortactin antibody.
  • the amount of a cortactin-associated transcript is measured by contacting the sample with a polynucleotide, e.g. a nucleic acid probe, that selectively hybridizes to the cortactin transcript.
  • a polynucleotide e.g. a nucleic acid probe
  • analysing the expression of cortactin may involve determining the nature of at least one expression product of a gene encoding cortactin in the presence of the candidate compound, wherein a change in the nature of the expression product in the presence of the compound when compared to the absence of the compound indicates that the compound is a potential modulator of cell proliferation.
  • the expression product analysed in this method may be, for example, mRNA or a protein.
  • the expression product is mRNA and the method involves detecting alternative spliced forms of an mRNA transcript when expression of the polynucleotide occurs in the presence of the candidate compound.
  • the expression product is a cortactin protein and the method involves detecting a change in the size and/or amino acid sequence of the protein produced when expression of the gene occurs in the presence of the candidate compound.
  • the analysis of cortactin expression may be achieved by exposing a translation system capable of expressing cortactin to the candidate compound and analysing the expression of cortactin in the presence and absence of the compound.
  • the translation system may be a cell-free translation system.
  • the translation system may comprise eukaryotic or prokaryotic cells.
  • An example of a screening method based on the analysis of cortactin expression may involve the following steps:
  • the cells used in this screening method may be any cells that can express cortactin, irrespective of the difference between natural and recombinant genes. Moreover, the derivation of the cortactin is not particularly limited.
  • the cells may be human derived, or may derive from mammals other than humans such as mice, or from other organisms. Transformed cells that contain expression vectors comprising nucleic acid sequences that encode cortactin may also be used.
  • the conditions for allowing the candidate compound to come into contact with the cells that can express cortactin are not limited, but it is preferable to select from among culture conditions (temperature, pH, culture composition, etc.) which will not kill the applicable cells, and in which the EMS-I gene can be expressed.
  • the term “reduced” refers not only the comparison with the control amount of expression, but also encompasses cases where no cortactin is expressed at all. Specifically, this includes circumstances wherein the amount of expression of cortactin is substantially zero.
  • the amount of expression of cortactin mRNA may be measured utilizing DNA array or well-known methods such as the Northern blot method, as well as the RT-PCR method that utilizes oligonucleotides having nucleotide sequences complementary to the nucleotide sequence of the applicable cortactin mRNA.
  • the amount of cortactin protein may be measured by implementing such well-known methods as the Western blot method utilizing an anti-cortactin antibody.
  • the method of the present invention involves analysing the activity of cortactin in the presence of a candidate compound.
  • analysing the activity of cortactin involves determining the ability of the candidate compound to modulate the binding of cortactin to a cortactin binding partner, wherein an altered level of binding of cortactin to the cortactin binding partner in the presence of the compound when compared to the absence of the compound indicates that the compound is a potential modulator of cell proliferation.
  • a reduced level of binding of cortactin to the cortactin binding partner in the presence of the compound indicates that the compound is an antiproliferative agent.
  • an increased level of binding of cortactin to the cortactin binding partner in the presence of the compound indicates that the compound is an agent that enhances cell proliferation.
  • the cortactin binding partner is selected from the group consisting of:
  • CD2AP (Daly, Biochem J. 382:13-25 (2004));
  • one of the cortactin binding partners e.g Arp2/3, F-actin or CD2AP is immobilized on a solid matrix, such as, for example an array of polymeric pins or a glass support.
  • the immobilized binding partner is a fusion polypeptide comprising Glutathione-S-transferase (GST), wherein the GST moiety facilitates immobilization of the protein to the solid phase support.
  • GST Glutathione-S-transferase
  • the second binding partner e.g. cortactm
  • the second binding partner in solution is brought into physical relation with the immobilized protein to form a protein complex, which complex is detected using antibodies directed against the second binding partner.
  • the antibodies are generally labelled with fluorescent molecules or conjugated to an enzyme (e.g. horseradish peroxidase), or alternatively, a second labelled antibody can be used that binds to the first antibody.
  • the second binding partner is expressed as a fusion polypeptide with a FLAG or oligo-histidine peptide tag, or other suitable immunogenic peptide, wherein antibodies against the peptide tag are used to detect the binding partner.
  • oligo-HIS tagged protein complexes can be detected by their binding to nickel-NTA resin (Qiagen), or FLAG-labeled protein complexes detected by their binding to FLAG M2 Affinity Gel (Kodak). It will be apparent to the skilled person that the assay format described herein is amenable to high throughput screening of samples, such as, for example, using a microarray of bound peptides or fusion proteins.
  • a two-hybrid assay as described in US Patent No. 6,316,223 may also be used to identify compounds that interfere with the binding of cortactm to one of its binding partners.
  • the basic mechanism of this system is similar to the yeast two hybrid system.
  • the binding partners are expressed as two distinct fusion proteins in a mammalian host cell.
  • a first fusion protein consists of a DNA binding domain which is fused to one of the binding partners
  • a second fusion protein consists of a transcriptional activation domain fused to the other binding partner.
  • the DNA binding domain binds to an operator sequence which controls expression of one or more reporter genes.
  • the transcriptional activation domain is recruited to the promoter through the functional interaction between binding partners. Subsequently, the transcriptional activation domain interacts with the basal transcription machinery of the cell, thereby activating expression of the reporter gene(s), the expression of which can be determined.
  • Candidate compounds that modulate the protein-protein interaction between the binding partners are identified by their ability to modulate transcription of the reporter gene(s) when incubated with the host cell. Antagonists will prevent or reduce reporter gene expression, while agonists will enhance reporter gene expression. In the case of small molecule modulators, these are added directly to the cell medium and reporter gene expression determined.
  • peptide modulators are expressible from nucleic acid that is transfected into the host cell and reporter gene expression determined. In fact, whole peptide libraries can be screened in transfected cells.
  • reverse two hybrid screens such as, for example, described by Vidal et al., Proc. Natl Acad. Sci USA 93, 10315-10320, 1996, may be employed to identify antagonist molecules.
  • Reverse hybrid screens differ from forward screens described above in so far as they employ a counter-selectable reporter gene, such as for example, CYH2 or LYS2, to select against the protein-protein interaction.
  • CYH2 or LYS2 a counter-selectable reporter gene
  • Cell survival or growth is reduced or prevented in the presence of a non-toxic substrate of the counter- selectable reporter gene product, which is converted by said gene product to a toxic compound. Accordingly, cells in which the protein-protein interaction of the invention does not occur, such as in the presence of an antagonist of said interaction, survive in the presence of the substrate, because it will not be converted to the toxic product.
  • a protein recruitment system such as that described in U.S. Patent No. 5, 776, 689 to Karin et al., may be used.
  • a protein-protein interaction is detected in a cell by the recruitment of an effector protein, which is not a transcription factor, to a specific cell compartment.
  • the effector protein Upon translocation of the effector protein to the cell compartment, the effector protein activates a reporter molecule present in that compartment, wherein activation of the reporter molecule is detectable, for example, by cell viability, indicating the presence of a protein-protein interaction.
  • the components of a protein recruitment system include a first expressible nucleic acid encoding a first fusion protein comprising a cortactin binding partner, and a second expressible nucleic acid molecule encoding a second fusion protein comprising a cell compartment localization domain and cortactin (or a portion thereof).
  • a cell line or cell strain in which the activity of an endogenous effector protein is defective or absent e.g. a yeast cell or other non-mammalian cell
  • the reporter molecule is not expressed.
  • a complex is formed between the fusion polypeptides as a consequence of the interaction between the binding partners, thereby directing translocation of the complex to the appropriate cell compartment mediated by the cell compartment localization domain (e.g. plasma membrane localization domain, nuclear localization domain, mitochondrial membrane localization domain, and the like), where the effector protein then activates the reporter molecule.
  • the cell compartment localization domain e.g. plasma membrane localization domain, nuclear localization domain, mitochondrial membrane localization domain, and the like
  • the effector protein then activates the reporter molecule.
  • Such a protein recruitment system can be practiced in essentially any type of cell, including, for example, mammalian, avian, insect and bacterial cells, and using various effector protein/reporter molecule systems.
  • a yeast cell based assay is performed, in which the interaction between cortactin and one or more of its binding partners results in the recruitment of a guanine nucleotide exchange factor (GEF or C3G) to the plasma membrane, wherein GEF or C3G activates a reporter molecule, such as Ras, thereby resulting in the survival of cells that otherwise would not survive under the particular cell culture conditions.
  • GEF or C3G guanine nucleotide exchange factor
  • Suitable cells for this purpose include, for example, Saccharomyces cerevisiae cdc25-2 cells, which grow at 36°C only when a functional GEF is expressed therein, Petitjean et al, Genetics 124, 797-806, 1990). Translocation of the GEF to the plasma membrane is facilitated by a plasma membrane localization domain.
  • Activation of Ras is detected, for example, by measuring cyclic AMP levels in the cells using commercially available assay kits and/or reagents.
  • duplicate incubations are carried out in the presence and absence of a test compound, or in the presence or absence of expression of a candidate antagonist peptide in the cell.
  • Reduced survival or growth of cells in the presence of a candidate compound or candidate peptide indicates that the peptide or compound is an antagonist of the interaction between cortactin and one or more of its binding partners.
  • a "reverse" protein recruitment system is also contemplated, wherein modified survival or modified growth of the cells is contingent on the disruption of the protein-protein interaction by the candidate compound or candidate peptide.
  • NIH 3T3 cells that constitutively express activated Ras in the presence of GEF can be used, wherein the absence of cell transformation is indicative of disruption of the protein complex by a candidate compound or peptide.
  • NIH 3T3 cells that constitutively express activated Ras in the presence of GEF have a transformed phenotype (Aronheim et al, Cell. 78, 949-961 , 1994).
  • small molecules are tested for their ability to interfere with binding of cortactin to one of its binding partners, by an adaptation of plate agar diffusion assay described by Vidal and Endoh, TIBS 17, 374-381, 1999, which is incorporated herein by reference.
  • analysing the activity of cortactin involves determining the ability of the candidate compound to modulate the cortactin-mediated actin polymerisation, wherein an altered level or rate of cortactin-mediated actin polymerisation in the presence of the compound indicates that the compound is a potential modulator of cell proliferation.
  • a reduced level or rate of cortactin-mediated actin polymerisation in the presence of the compound indicates that the compound is a potential antiproliferative agent.
  • an increased level or rate of cortactin-mediated actin polymerisation in the presence of the compound indicates that the compound is an agent that enhances cell proliferation.
  • the ability of a compound to modulate cortactin- mediated actin polymerisation can be measured in an in vitro actin polymerisation assay, wherein the assay contains cortactin and other molecules and reagents required for actin polymerization, such as, but not limited to G-actin and Arp2/3.
  • the assay contains cortactin and other molecules and reagents required for actin polymerization, such as, but not limited to G-actin and Arp2/3.
  • Such assays are know to those skilled in the art (for example, see Urono et al. (2001) Activation of Arp2/3 complex-mediated actin polymerization by cortactin. Nat Cell Biol. 3: 259-266) and can be adapted for use in high-throughput screening for small-molecule modulators of a target proteins activity (for example, see Peterson et al. (2001) A chemical inhibitor of N-WASP reveals a new mechanism for targeting protein interactions. PNAS. 98: 10624-
  • the present invention also provides a candidate compound identified by a screening method of the present invention.
  • the candidate compound is selected from the group consisting of a peptide, such as a peptide derived from cortactin, a cortactin dominant-negative mutant, an antibody directed against cortactin, non-peptide inhibitors of cortactin such as small organic molecules, antisense compounds directed against cortactin-encoding mRNA, anti-cortactin catalytic molecules such as ribozymes or a ⁇ -DNAzymes and dsRNA, for example RNAi molecules, that target cortactin expression.
  • a peptide such as a peptide derived from cortactin, a cortactin dominant-negative mutant
  • an antibody directed against cortactin such as small organic molecules
  • antisense compounds directed against cortactin-encoding mRNA anti-cortactin catalytic molecules
  • anti-cortactin catalytic molecules such as ribozymes or a ⁇ -DNAzymes and dsRNA, for example RNAi molecules, that target cor
  • the candidate compound may be obtained from expression products of a gene library, a low molecular weight compound library (such as the low molecular weight compound library of ChemBridge Research Laboratories), a cell extract, microorganism culture supernatant, bacterial cell components and the like.
  • a low molecular weight compound library such as the low molecular weight compound library of ChemBridge Research Laboratories
  • a cell extract such as the low molecular weight compound library of ChemBridge Research Laboratories
  • microorganism culture supernatant such as the low molecular weight compound library of ChemBridge Research Laboratories
  • the candidate compound is a protein.
  • protein in this context it is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides.
  • the protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures.
  • amino acid or “peptide residue”, as used herein means both naturally occurring and synthetic amino acids. For example, homophenylalanine, citrulline and noreleucine are considered amino acids for the purposes of the invention.
  • Amino acid also includes imino acid residues such as proline and hydroxyproline.
  • the side chains may be in either the (R) or the (S) configuration. In the preferred embodiment, the amino acids are in the (S) or (L)-configuration. If non-naturally occurring side chains are used, non-amino acid substituents may be used, for example to prevent or retard in vivo degradations.
  • the candidate compound is a naturally occurring protein or fragment of a naturally occurring protein.
  • cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts may be used. In this way libraries of prokaryotic and eukaryotic proteins may be made.
  • Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred.
  • the candidate compounds are peptides of from about 5 to about 30 amino acids, with from about 5 to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred.
  • the peptides may be digests of naturally occurring proteins, random peptides, or "biased” random peptides.
  • randomized or grammatical equivalents herein is meant that each peptide consists of essentially random amino acids. Since generally these random peptides are chemically synthesized, they may incorporate any amino acid at any position.
  • the synthetic process can be designed to generate randomized proteins to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous compounds.
  • combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka (1991) Int. J. Pept. Prot. Res., 37: 487-493, Houghton et a (1991) Nature, 354: 84-88).
  • Peptide synthesis is by no means the only approach envisioned and intended for use with the present invention.
  • Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (PCT Publication No WO 91/19735, Dec.
  • nucleic acid libraries nucleic acid libraries
  • peptide nucleic acid libraries see, e.g., U.S. Pat. No. 5,539,083
  • antibody libraries see, e.g., Vaughn et al (1996) Nature Biotechnology, 14(3): 309-314), and PCT/US96/10287)
  • carbohydrate libraries see, e.g., Liang et al (1996) Science, 274: 1520-1522, and U.S. Pat. No. 5,593,853
  • small organic molecule libraries see, e.g., benzodiazepines, Baum (1993) C&EN, Jan 18, page 33, isoprenoids U.S. Pat. No.
  • cortactin fragments such as for example peptidyl cortactin inhibitors, are chemically or recombinantly synthesized as oligopeptides
  • cortactin fragments are produced by digestion of native or recombinantly produced cortactin by, for example, using a protease, e.g., trypsin, thermolysin, chymotrypsin, or pepsin.
  • a protease e.g., trypsin, thermolysin, chymotrypsin, or pepsin.
  • Computer analysis using commercially available software, e.g. MacVector, Omega, PCGene, Molecular Simulation, Inc.
  • the proteolytic or synthetic fragments can comprise as many amino acid residues as are necessary to partially or completely inhibit cortactin function.
  • Preferred fragments will comprise at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length.
  • Proteins or peptides may also be dominant-negative mutants of cortactin.
  • the term "dominant-negative mutant” refers to a cortactin polypeptide that has been mutated from its natural state and that interacts with a protein that cortactin normally interacts with thereby preventing endogenous native cortactin from forming the interaction.
  • the candidate compound is an anti-cortactin antibody.
  • antibody as used in this invention includes intact molecules as well as fragments thereof, such as Fab, F(ab')2, and Fv which are capable of binding an epitopic determinant of cortactin. These antibody fragments retain some ability to selectively bind with its antigen and are defined as follows:
  • Fab the fragment which contains a monovalent antigen-binding fragment of an antibody molecule can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;
  • Fab' the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;
  • (Fab')2 the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction
  • F(ab)2 is a dimer of two Fab' fragments held together by two disulfide bonds
  • Fv defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains
  • Single chain antibody defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.
  • Antibodies of the present invention can be prepared using intact cortactin or fragments thereof as the immunizing antigen.
  • a peptide used to immunize an animal can be derived from translated cDNA or chemical synthesis and is purified and conjugated to a carrier protein, if desired.
  • carrier protein e.g., a carrier protein
  • Such commonly used carriers which are chemically coupled to the peptide include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • the coupled peptide may then be used to immunize the animal (e.g., a mouse or a rabbit).
  • polyclonal antibodies can be further purified, for example, by binding to and elution from a matrix to which the peptide to which the antibodies were raised is bound.
  • a matrix to which the peptide to which the antibodies were raised is bound.
  • Those of skill in the art will know of various techniques common in the immunology arts for purification and/or concentration of polyclonal antibodies, as well as monoclonal antibodies (See for example, Coligan, et ah, Unit 9, Current Protocols in Immunology, Wiley Interscience, 1991, incorporated by reference).
  • Monoclonal antibodies may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture, such as, for example, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler et ⁇ l. Nature 256, 495-497, 1975; Kozbor et al, J. Immunol. Methods 81, 31-42, 1985; Cote et al, Proc. Natl. Acad. Sci. USA 80, 2026- 2030, 1983; Cole et al, MoI. Cell Biol. 62, 109-120, 1984).
  • bacteriophage lambda vector system This vector system has been used to express a combinatorial library of Fab fragments from the mouse antibody repertoire in Escherichia coli (Huse, et ah, Science, 246:1275-1281, 1989) and from the human antibody repertoire (Mullinax, et ah, Proc. Nat. Acad. Sci., 87:8095-8099, 1990).
  • Hybridomas which secrete a desired monoclonal antibody can be produced in various ways using techniques well understood by those having ordinary skill in the art and will not be repeated here. Details of these techniques are described in such references as Monoclonal Antibodies-Hybridomas: A New Dimension in Biological Analysis, Edited by Roger H. Kennett, et ah, Plenum Press, 1980; and U.S. 4,172,124, incorporated by reference.
  • chimeric antibody molecules with various combinations of "humanized” antibodies include combining murine variable regions with human constant regions (Cabily, et a Proc. Natl. Acad. Sci. USA, 81:3273, 1984), or by grafting the murine-antibody complementarity determining regions (CDRs) onto the human framework (Riechmann, et ah, Nature 332:323, 1988).
  • CDRs murine-antibody complementarity determining regions
  • the candidate compound is an antisense compound.
  • antisense compounds encompasses DNA or RNA molecules that are complementary to at least a portion of a cortactin mRNA molecule (Izant and
  • Antisense oligomers complementary to at least about 15 contiguous nucleotides of cortactin-encoding mRNA are preferred, since they are easily synthesized and are less likely to cause problems than larger molecules when introduced into the target cortactin-producing cell.
  • the use of antisense methods is well known in the art (Marcus-Sakura, Anal. Biochem. 172: 289, 1988).
  • Preferred antisense nucleic acid will comprise a nucleotide sequence that is complementary to about at least 15 contiguous nucleotides of a sequence encoding the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:4.
  • the candidate compound is a catalytic nucleic acid.
  • catalytic nucleic acid refers to a DNA molecule or DNA-containing molecule (also known in the art as a "DNAzyme”) or an RNA or RNA-containing molecule (also known as a "ribozyme”) which specifically recognizes a distinct substrate and catalyzes the chemical modification of this substrate.
  • the nucleic acid bases in the catalytic nucleic acid can be bases A 5 C, G, T and U, as well as derivatives thereof. Derivatives of these bases are well known in the art.
  • the catalytic nucleic acid contains an antisense sequence for specific recognition of a target nucleic acid, and a nucleic acid cleaving enzymatic activity (also referred to herein as the "catalytic domain").
  • a nucleic acid cleaving enzymatic activity also referred to herein as the "catalytic domain"
  • preferred ribozymes and DNAzymes will comprise a nucleotide sequence that is complementary to at least about 12-15 contiguous nucleotides of a sequence encoding the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO:4.
  • ribozymes that are particularly useful in this invention are the hammerhead ribozyme (Haseloff and Gerlach, Nature, 334: 585 (1988), Perriman et ah, Gene, 133: 157 (1992)) and the hairpin ribozyme (Shippy et ah, MoI. Biotech. 12: 117 (1999)).
  • the ribozymes of this invention and DNA encoding the ribozymes can be chemically synthesized using methods well known in the art.
  • the ribozymes can also be prepared from a DNA molecule (that upon transcription, yields an RNA molecule) operably linked to an RNA polymerase promoter, e.g., the promoter for T7 RNA polymerase or SP6 RNA polymerase.
  • an RNA polymerase promoter e.g., the promoter for T7 RNA polymerase or SP6 RNA polymerase.
  • a nucleic acid molecule i.e., DNA or cDNA, coding for the ribozymes of this invention.
  • the ribozyme can be produced in vitro upon incubation with RNA polymerase and nucleotides.
  • the DNA can be inserted into an expression cassette or transcription cassette.
  • the RNA molecule can be modified by ligation to a DNA molecule having the ability to stabilize the ribozyme and make it resistant to RNase.
  • the ribozyme can be modified to the phosphothio analog for use in liposome delivery systems. This modification also renders the ribozyme resistant to endonuclease activity.
  • the candidate compound is an RNA inhibitor.
  • dsRNA is particularly useful for specifically inhibiting the production of a particular protein.
  • Dougherty and Parks have provided a model for the mechanism by which dsRNA can be used to reduce protein production.
  • This model has recently been modified and expanded by Waterhouse et al. (Proc. Natl. Acad. Sci. 95: 13959 (1998)).
  • This technology relies on the presence of dsRNA molecules that contain a sequence that is essentially identical to the mRNA of the gene of interest, in this case an mRNA encoding cortactin.
  • the dsRNA can be produced in a single open reading frame in a recombinant vector or host cell, where the sense and anti-sense sequences are flanked by an unrelated sequence which enables the sense and anti-sense sequences to hybridize to form the dsRNA molecule with the unrelated sequence forming a loop structure.
  • the design and production of suitable dsRNA molecules targeted against cortactin is well within the capacity of a person skilled in the art, particularly considering Dougherty and Parks (1995, supra), Waterhouse et al. (1998, supra), WO 99/32619, WO 99/53050, WO 99/49029, and WO 01/34815.
  • RNAi refers to homologous double stranded RNA (dsRNA) that specifically targets a gene product, thereby resulting in a null or hypomorphic phenotype.
  • small interfering RNA refers to short nucleotide dsRNA and as such is a subset of RNAi.
  • the dsRNA comprises two nucleotide sequences, in the case of siRNA two short nucleotide sequences, derived from the target RNA encoding cortactin and having self-complementarity such that they can anneal, and interfere with expression of a target gene, presumably at the post- transcriptional level.
  • RNAi molecules are described by Fire et ah, Nature 391, 806- 811, 1998, and reviewed by Sharp, Genes & Development, 13, 139-141, 1999).
  • shRNA short hairpin RNA
  • siRNA comprises a single strand of nucleic acid wherein the complementary sequences are separated by an intervening hairpin loop such that, following introduction to a cell, it is processed by cleavage of the hairpin loop into siRNA. Accordingly, each and every embodiment described herein as being applicable to siRNA is equally applicable to shRNA.
  • DNA-directed RNA interference relies on DNA templates containing a promoter for the expression of RNAi in mammalian cells.
  • the promoter directs the synthesis of RNA transcripts whose 3' ends are defined by a termination sequence, typically a stretch of 4-5 thymidines. This allows the use of DNA templates to synthesize, in vivo, small RNA duplexes that are structurally equivalent to active RNAi 5 and in particular siRNA, synthesized in vitro.
  • dsRNA forms in the cell and leads to the degradation of the target mRNA.
  • RNA agent refers to an RNA sequence that elicits RNAi; and the term “ddRNAi agent” refers to an RNAi agent that is transcribed e.g. from a suitable DNA vector. In some embodiments of the present invention, ddRNAi agents are expressed initially as shRNAs.
  • RNAi expression cassette refers to a cassette according to embodiments of the present invention having at least one [promoter-sequence to be expressed encoding an RNAi agent-terminator] unit.
  • multiple promoter RNAi expression cassette refers to an RNAi expression cassette comprising two or more [promoter- sequence to be expressed encoding an RNAi agent-terminator] units.
  • RNAi expression construct or "RNAi expression vector” refer to vectors containing an RNAi expression cassette.
  • Preferred ddRNAi expression cassettes most often will include at least one promoter, a sequence to be expressed encoding an RNAi agent, and at least one terminator.
  • the ddRNAi expression cassette may be ligated, for example, into viral delivery vector, and the ddRNAi viral delivery vector may be packaged into viral particles. Finally, viral particles may be delivered to target cells, tissues or organs. Alternatively, the ddRNAi expression cassette is ligated into a non- viral delivery vector which is then delivered to target cells, tissues or organs.
  • the siRNA agent may be constructed specifically for delivery, such that the siRNA is formulated with an appropriate carrier for delivery and the siRNA agent/carrier is delivered to target cells, tissues, or organs.
  • RNAi agents according to the present invention are generated synthetically or enzymatically by a number of different protocols known to those skilled in the art and purified using standard recombinant DNA techniques as described in, for example, Sambrook et ah, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (1989), and under regulations described in, e.g., United States Dept. of HHS 5 National Institute of Health (NIH) Guidelines for Recombinant DNA Research.
  • NASH National Institute of Health
  • RNAi agents may comprise either synthetic small-interfering (si)RNAs or DNA- directed RNAs (ddRNAs).
  • Synthetic siRNAs may be manufactured by methods known in the art such as by typical oligonucleotide synthesis, and often will incorporate chemical modifications to increase half life and/or efficacy of the siRNA agent, and/or to allow for a more robust delivery formulation.
  • Many modifications of oligonucleotides are known in the art.
  • US Serial No. 6,620,805 discloses an oligonucleotide that is combined with a macrocycle having a net positive charge such as a porphyrin
  • US Serial No. 6,673,611 discloses various formulas
  • oligomers that include a modification comprising a polycyclic sugar surrogate; such as a cyclobutyl nucleoside, cyclopentyl nucleoside, proline nucleoside, cyclohexene nucleoside, hexose nucleoside or a cyclohexane nucleoside; and oligomers that include a non-phosphorous-containing internucleoside linkage; US Pub. No 2004/0171579 discloses a modified oligonucleotide where the modification is a 2' substituent group on a sugar moiety that is not H or OH; US Pub. No.
  • 2004/0171030 discloses a modified base for binding to a cytosine, uracil, or thymine base in the opposite strand comprising a boronated C and U or T modified binding base having a boron-containing substituent selected from the group consisting of-BH2CN, -BH3, and --BH2COOR, wherein R is Cl to Cl 8 alkyl;
  • US Pub. No. 2004/0161844 discloses oligonucleotides having phosphoramidate internucleoside linkages such as a 3'aminophosphoramidate, aminoalkylphosphor- amidate, or aminoalkylphosphorthioamidate internucleoside linkage; US Pub. No.
  • 2004/0161844 discloses yet other modified sugar and/or backbone modifications, where in some embodiments, the modification is a peptide nucleic acid, a peptide nucleic acid mimic, a morpholino nucleic acid, hexose sugar with an amide linkage, cyclohexenyl nucleic acid (CeNA), or an acyclic backbone moiety;
  • US Pub. No. 2004/0161777 discloses oligonucleotides with a 3' terminal cap group; US Pub. No.
  • 2004/0147470 discloses oligomeric compounds that include one or more cross-linkages that improve nuclease resistance or modify or enhance the pharmacokinetic and phamacodynamic properties of the oligomeric compound where such cross-linkages comprise a disulfide, amide, amine, oxime, oxyamine, oxyimine, morpholino, thioether, urea, thiourea, or sulfonamide moiety; US Pub. No.
  • 2004147023 discloses a gapmer comprising two terminal RNA segments having nucleotides of a first type and an internal RNA segment having nucleotides of a second type where nucleotides of said first type independently include at least one sugar substituent where the sugar substituent comprises a halogen, amino, trifluoroalkyl, trifluoroalkoxy, azido, aminooxy, alkyl, alkenyl, alkynyl, O-, S-, or N(R*)-alkyl; O-, S-, or N(R*)-alkenyl; O-, S- or N(R*)-alkynyl; O-, S- or N-aryl, O-, S-, or N(R*)-aralkyl group; where the alkyl, alkenyl, alkynyl, aryl or aralkyl may be a substituted or unsubstituted alkyl, alkenyl, alkynyl, a
  • ddRNAi agents DNA-directed RNAi agents may be employed.
  • ddRNAi agents are obtained using an expression cassette, most often containing at least one promoter, at least one ddRNAi sequence and at least one terminator in a viral or non- viral vector backbone.
  • Promoters of variable strength may be employed. For example, use of two or more strong promoters (such as a Pol Ill-type promoter) may tax the cell, by, e.g., depleting the pool of available nucleotides or other cellular components needed for transcription. In addition or alternatively, use of several strong promoters may cause a toxic level of expression of RNAi agents in the cell. Thus, in some embodiments one or more of the promoters in the multiple-promoter RNAi expression cassette may be weaker than other promoters in the cassette, or all promoters in the cassette may express RNAi agents at less than a maximum rate. Promoters also may or may not be modified using molecular techniques, or otherwise, e.g., through regulation elements, to attain weaker levels of transcription.
  • Promoters useful in some embodiments of the present invention may be tissue-specific or cell-specific.
  • tissue specific refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest to a specific type of tissue (e.g., epithelial tissue) in the relative absence of expression of the same nucleotide sequence of interest in a different type of tissue (e.g., muscle).
  • cell-specific refers to a promoter which is capable of directing selective expression of a nucleotide sequence of interest in a specific type of cell in the relative absence of expression of the same nucleotide sequence of interest in a different type of cell within the same tissue (see, e.g., Higashibata, et al, J. Bone Miner. Res. Jan 19(l),78-88, 2004; Hoggatt, et al, Circ. Res., Dec. 91(12), 1151-59, 2002; Sohal, et al, Circ. Res. M 89(1), 20-25, 2001; and Zhang, et al, Genome Res. Jan 14(1), 79-89, 2004).
  • cell-specific when applied to a promoter also means a promoter capable of promoting selective expression of a nucleotide sequence of interest in a region within a single tissue.
  • promoters may be constitutive or regulatable. Additionally, promoters may be modified so as to possess different specificities.
  • constitutive when made in reference to a promoter means that the promoter is capable of directing transcription of an operably linked nucleic acid sequence in the absence of a specific stimulus (e.g., heat shock, chemicals, light, etc.). Typically, constitutive promoters are capable of directing expression of a coding sequence in substantially any cell and any tissue.
  • the promoters used to transcribe the RNAi agents preferably are constitutive promoters, such as the promoters for ubiquitin, CMV, ⁇ - actin, histone H4, EF-lalfa or pgk genes controlled by RNA polymerase II, or promoter elements controlled by RNA polymerase I.
  • a Pol II promoter such as CMV, SV40, Ul, ⁇ -actin or a hybrid Pol II promoter is employed.
  • promoter elements controlled by RNA polymerase III are used, such as the U6 promoters (U6-1, U6-8, U6-9, e.g.), Hl promoter, 7SL promoter, the human Y promoters (hYl, hY3, hY4 (see Maraia, et al, Nucleic Acids Res 22(15), 3045-52, 1994) and hY5 (see Maraia, et al, Nucleic Acids Res 24(18), 3552-59, 1994), the human MRP-7-2 promoter, Adenovirus VAl promoter, human tRNA promoters, the 5s ribosomal RNA promoters, as well as functional hybrids and combinations of any of these promoters.
  • promoters that allow for inducible expression of the RNAi agent.
  • a number of systems for inducible expression using such promoters are known in the art, including but not limited to the tetracycline responsive system and the lac operator-repressor system (see WO 03/022052 Al; and US 2002/0162126 Al), the ecdysone regulated system, or promoters regulated by glucocorticoids, progestins, estrogen, RU-486, steroids, thyroid hormones, cyclic AMP, cytokines, the calciferol family of regulators, or the metallothionein promoter (regulated by inorganic metals).
  • Particularly preferred promoters for expression in mammalian cells that express cortactin include the CMV promoter, ubiquitin promoter, U6 small nuclear RNA promoter (Lee et ah, Nature Biotech. 20, 500-505, 2002; Miyagishi et a;., Nature
  • RNA polymerase III promoter (Brummelkamp et al., Science 296, 550-553, 2002), or other RNA polymerase III promoter.
  • the pol III terminator is also preferred for such applications.
  • Other promoters and terminators are not to be excluded.
  • Preferred siRNA molecules comprise a nucleotide sequence that is identical to about 19-21 contiguous nucleotides of the target mRNA.
  • the target sequence in cortactin mRNA commences with the dinucleotide AA, comprises a GC-content of about 30-70% (preferably, 30-60%, more preferably 40-60% and more preferably about 45%-55%), and does not have a high percentage identity to any nucleotide sequence other than cortactin in the genome of the animal in which it is to be introduced, e.g., as determined by standard BLAST search.
  • the siRNA is preferably capable of downregulating expression of human cortactin in a cell.
  • this should not be taken to indicate a requirement for the siRNA to be specific for human cortactin-encoding genes.
  • the siRNA molecules it is possible and appropriate in certain circumstances for the siRNA molecules to reduce expression of both endogenous murine cortactin, as well as ectopically expressed human cortactin in the cell.
  • Preferred siRNA against a cortactin encoding region comprises a sequence set forth in any one of SEQ ID Nos: 5-28.
  • the candidate compound is a small molecule inhibitor.
  • organic molecules may be assayed for their ability to modulate the immune system.
  • suitable organic molecules may be selected either from a chemical library, wherein chemicals are assayed individually, or from combinatorial chemical libraries where multiple compounds are assayed at once, then deconvolved to determine and isolate the most active compounds.
  • combinatorial chemical libraries include those described by Agrafiotis et al, "System and method of automatically generating chemical compounds with desired properties," U.S. Pat. No. 5,463,564; Armstrong, R. W., "Synthesis of combinatorial arrays of organic compounds through the use of multiple component combinatorial array syntheses," WO 95/02566; Baldwin, J. J. et al, “Sulfonamide derivatives and their use," WO 95/24186; Baldwin, J. J. et al, “Combinatorial dihydrobenzopyran library," WO 95/30642; Brenner, S., “New kit for preparing combinatorial libraries.” WO 95/16918; Chenera, B.
  • the present invention involves screening small molecule chemodiversity represented within libraries of parent and fractionated natural product extracts, to detect bioactive compounds as potential candidates for further characterization.
  • the process of primary screening may involve the use of specialised assay technologies, coupled with automated systems, which allow test sample throughputs of up to 50,000 per day.
  • the screening process may involve the use of a robotic screening system comprising a precise, six-axis robotic arm mounted on a linear track. Such as system links to all instrumentation and hardware, allowing microtiter plates to be transferred to any location on the system.
  • Hardware includes plate carousels for storage and access of sample and assay plates, an automated system with robotic arm for liquid handling, a platewise microplate pipetting system, a plate shaker and a plate washer.
  • This system also has a plate reader capable of fluorometric, photometric and luminometric detection.
  • a number of stand-alone instruments for rapid microplate pipetting and for detection of a variety of signal read-outs from assay plates may be employed.
  • the process of dereplication may be used to select a small sub-population of hits identified in a primary screen that are most likely to contain active compounds with the desired characteristics.
  • dereplication is an important success-determining and rate-limiting step in natural products drug discovery.
  • Dereplication may be performed as follows. All hits from the initial cortactin inhibitor screen are subjected to a high-capacity fractionation procedure designed to generate information about the relative polarity of all active compounds present. Based on this information, all extracts displaying bioactivity in one or more of these initial fractions are progressed for HPLC separations using short gradients tailored to provide high resolution over the appropriate polarity ranges. With coupled UV/visible detection of eluates, testing of fractions for bioactivity both in the primary screen and relevant secondary assays, and analysis of active fractions by LC-MS, a package of physicochemical and bioactivity data on pure or nearly pure active HPLC fractions from all screen hits is generated.
  • Prioritized screen hits emerging from the de-replication process may be progressed for isolation and full chemical characterization of the active compounds present.
  • scaled-up quantities of the appropriate extracts may be first prepared by re-fermenting the producing organisms.
  • plant tissue extracts there are sufficient stocks of most of the dried and ground plant tissue specimens to prepare further quantities of extract for chemical isolation work.
  • the chemical isolation program aims to purity enough of each active compound to conduct structure elucidation work and further profiling of biological activity (typically 2 - 20 mg). Structures may be determined primarily on the basis of mass spectrometry (MS) and nuclear magnetic resonance (NMR) data.
  • Preferred bioassays developed as primary screens are also backed up by several secondary assays designed to detect false hits (eg due to interference with the assay detection system), hits due to unrelated modes of action (eg cytotoxicity in functional cell-based screens) or hits that fail to show the desired profile of biological specificity.
  • Most secondary assays are reserved for use at a late stage in the dereplication process when they can be applied to pure or nearly pure active fractions derived from the hit extracts identified in primary screens.
  • Dose response inhibition of lead hits identified in the cortactin assay are preferably also performed in order to measure potency and efficacy of inhibition.
  • Compounds identified by the screening methods of the present invention may be validated by in vitro or in vivo cell proliferation assays.
  • reproductive assays can be used to determine the number of cells in a culture that are capable of forming colonies in vitro. In these types of experiments, cells are plated at low densities and the number of colonies is scored after a growth period. These clonogenic assays are reliable methods for assaying viable cell numbers. However, they are time-consuming and may become impractical when many samples have to be analyzed.
  • Alternative cell proliferation assays are direct proliferation assays that use DNA synthesis as an indicator of cell growth. The close association between DNA synthesis and cell doubling makes the measurement of DNA synthesis attractive for assessing cell proliferation. If labeled DNA precursors are added to the cell culture, cells that are about to divide incorporate the labeled nucleotide into their DNA. Traditionally, those assays involve the use of radiolabeled nucleosides, particularly tritiated thymidine ([ 3 H]-TdR). The amount of [ 3 H]-TdR incorporated into the cellular DNA is quantitated by liquid scintillation counting (LSC).
  • LSC liquid scintillation counting
  • thymidine analogue 5-bromo-2'-deoxy-uridine (BrdU) is incorporated into cellular DNA like thymidine.
  • the incorporated BrdU can be detected by a quantitative cellular enzyme immunoassay using monoclonal anti- bodies directed against BrdU.
  • the use of BrdU for cell proliferation assays circumvents the disadvantages associated with radioactive compounds such as [ 3 H]- TdR.
  • a sensitive cell proliferation assay utilizing Calcein-AM a fluorescent probe for staining viable cells (excitation.: ⁇ 485 nm; emission: -520 nm) has also been developed by Calbiochem.
  • the amount of fluorescent dye produced by cellular esterases is proportional to the number of viable cells.
  • the detection range is from 100 to 30,000 cells.
  • This "Utrasensitive Cell Proliferation Assay Kit” is especially suitable for the fast and convenient determination of viable cell numbers in cell proliferation studies.
  • the reduction of tetrazolium salts is also recognized as a safe, accurate alternative to radiometric testing.
  • the yellow tetrazolium salt (MTT) is reduced in metabolically active cells to form insoluble purple formazan crystals, which are solubilized by the addition of a detergent.
  • the color can then be quantified by spectrophotometric means. For each cell type a linear relationship between cell number and absorbance is established, enabling accurate, straightforward quantification of changes in proliferation. This type of assay is exemplified herein in Example 1.
  • Compounds can be also assayed for their ability to inhibit proliferation of cancer cells when they are grown as tumours in nude mice. For example, many cancer cell lines form tumours when injected subcutaneously into the upper back region of Balb/c athymic nude mice. Tumour growth rates can be determined by direct assessment of tumour size using calipers. Compounds can be administered to the tumour-bearing animals via a number of routes, eg orally, subcutaneously, intravenously or intraperitoneally. Tumour sections can be isolated and analysed for cell proliferation rates eg by Ki67 staining.
  • the methods of the present invention provide a rational method for designing and selecting compounds including antibodies which interact with and modulate the activity of cortactin. In the majority of cases these compounds will require further development in order to increase activity. It is intended that in particular embodiments the methods of the present invention includes such further developmental steps. For example, it is intended that embodiments of the present invention further include manufacturing steps such as incorporating the compound into a pharmaceutical composition in the manufacture of a medicament.
  • the method further comprises formulating the identified compound for administration to a human or a non-human animal as described herein.
  • the present invention clearly encompasses the use of any in silico analytical method and/or industrial process for carrying the screening methods described herein into a pilot scale production or industrial scale production of a compound identified in such screens.
  • This invention also provides for the provision of information for any such production.
  • the present invention also provides a process for identifying or determining a modulator of cell proliferation, said method comprising:
  • step (i) determination of the structure of the compound is implicit in step (i). This is because the skilled artisan will be aware of the name and/or structure of the compound at the time of performing the screen.
  • the term "providing the compound or modulator” shall be taken to include any chemical or recombinant synthetic means for producing said compound or modulator or alternatively, the provision or a compound or modulator that has been previously synthesized by any person or means.
  • the compound or modulator or the name or structure of the compound or modulator is provided with an indication as to its use e.g., as determined by a screen described herein.
  • the present invention also provides a process for producing a modulator of cell proliferation, said method comprising:
  • the synthesized compound or modulator or the name or structure of the compound or modulator is provided with an indication as to its use e.g., as determined by a screen described herein.
  • the present invention also provides a composition for inhibiting cell proliferation, the composition comprising an antiproliferative agent selected from the group consisting of a peptide derived from cortactin, a cortactin dominant-negative mutant, an antibody directed against cortactin, antisense compounds directed against cortactin-encoding mRNA, anti-cortactin catalytic molecules such as ribozymes or a DNAzymes, dsRNA and small interfering RNA (RNAi) molecules that target cortactin expression or combinations of any of these agents.
  • an antiproliferative agent selected from the group consisting of a peptide derived from cortactin, a cortactin dominant-negative mutant, an antibody directed against cortactin, antisense compounds directed against cortactin-encoding mRNA, anti-cortactin catalytic molecules such as ribozymes or a DNAzymes, dsRNA and small interfering RNA (RNAi) molecules that target cortactin expression or combinations of any
  • the composition comprises a small interfering RNA (RNAi) molecule that targets cortactin expression.
  • RNAi small interfering RNA
  • the RNAi molecule comprises a sequence as shown in any one of SEQ ID NOs: 5 to 29.
  • the present invention also provides a method for modulating cell proliferation, the method comprising administering to the cell population a compound that modulates the activity or expression of cortactin in an amount effective to modulate cell proliferation.
  • the compound specifically modulates the activity or expression of cortactin.
  • the method is directed at modulating cell proliferation in a subject in which a compound that modulates the activity or expression of cortactin is administered to the subject in an amount effective to modulate cell proliferation.
  • the present invention also provides a method for modulating cell proliferation in a subject, the method comprising administering to the subject a compound that modulates the activity or expression of cortactin in an amount effective to modulate cell proliferation.
  • cortactin By “specifically modulates the activity or expression of cortactin” we mean that the compound significantly modulates the activity or expression of cortactin without significantly modulating the activity or expression of other proteins in the subject or cell population.
  • the compound reduces or inhibits the activity or expression of cortactin in an amount effective to reduce or inhibit cell proliferation in the subject or cell population.
  • the compound is administered in an amount sufficient to reduce the activity or expression of cortactin to normal levels.
  • normal levels we mean the levels of activity or expression of cortactin that are typically found in the cells of healthy individuals.
  • the term "healthy individual” shall be taken to mean an individual who is known not to suffer from a hyperproliferative disorder and does not have elevated levels of cortactin expression or activity.
  • the present invention is particularly useful for the early treatment and prevention of cancer, it is preferred that the healthy individual is asymptomatic with respect to the early symptoms associated with a particular cancer.
  • this method comprises administering to a subject an antiproliferative agent selected from the group consisting of a peptide derived from cortactin, a cortactin dominant-negative mutant, an antibody directed against cortactin, antisense compounds directed against cortactin-encoding mRNA, anti-cortactin catalytic molecules such as ribozymes or DNAzymes, dsRNA and RNAi molecules (especially small interfering RNA (siRNA) molecules) that target cortactin expression or combinations of any of these agents. More preferably, the method comprises administering to the subject a RNAi molecule, suitably a siRNA molecule, that targets cortactin expression.
  • the RNAi molecule comprises a sequence as shown in any one of SEQ ID NOs: 5 to 29.
  • the compound increases the activity or expression of cortactin in an amount effective to induce cell proliferation in the subject or cell population.
  • this method involves administering to the cells or subject a compound that has been identified as being capable of increasing the activity or expression of cortactin.
  • this method involves manipulation of the cells of the cell population or of a subject to increase levels of expression of cortactin.
  • manipulation of the cells may involve genetic modification of the cortactin promoter region so as to increase expression of the cortactin gene.
  • the present invention also provides a method for treating or preventing a disorder associated with aberrant cell proliferation in a subject, the method comprising administering to the subject a compound that modulates the activity or expression of cortactin in an amount effective to treat or prevent the disorder.
  • the invention provides a method for treating or preventing a hyperproliferative disorder, the method comprising administering to the subject a compound that reduces or inhibits the activity or expression of cortactin in an amount effective to reduce or inhibit cell proliferation.
  • Suitable hyperproliferative disorders include cancer and psoriasis.
  • the invention provides a method for treating or preventing a condition that would benefit from increased cell proliferation, the method comprising administering to the subject a compound that enhances the activity of cortactin in an amount effective to treat the condition.
  • a compound that enhances the activity of cortactin in an amount effective to treat the condition.
  • conditions that would benefit from increased cell proliferation include wound healing, tissue or organ regeneration or rebuilding, and hematopoietic disorders such as hypoproliferative anemia.
  • the candidate compound is in the form of a low molecular weight compound, a peptide or a protein such as an antibody
  • the substance can be formulated into the ordinary pharmaceutical compositions (pharmaceutical preparations) which are generally used for such forms, and such compositions can be administered orally or parenterally.
  • pharmaceutical compositions pharmaceutical preparations
  • such compositions can be administered orally or parenterally.
  • dosage forms and methods of administration can be utilized.
  • the dosage form includes such representative forms as solid preparations, e.g. tablets, pills, powders, fine powders, granules, and capsules, and liquid preparations, e.g. solutions, suspensions, emulsions, syrups, and elixirs. These forms can be classified by the route of administration into said oral dosage forms or various parenteral dosage forms such as transnasal preparations, transdermal preparations, rectal preparations (suppositories), sublingual preparations, vaginal preparations, injections (intravenous, intraarterial, intramuscular, subcutaneous, intradermal) and drip injections.
  • solid preparations e.g. tablets, pills, powders, fine powders, granules, and capsules
  • liquid preparations e.g. solutions, suspensions, emulsions, syrups, and elixirs.
  • parenteral dosage forms such as transnasal preparations, transdermal preparations, rectal preparations (suppositories
  • the oral preparations may for example be tablets, pills, powders, fine powders, granules, capsules, solutions, suspensions, emulsions, syrups, etc. and the rectal and vaginal preparations include tablets, pills, and capsules, among others.
  • the transdermal preparations may not only be liquid preparations, such as lotions, but also be semi-solid preparations, such as reams, ointments, and so forth.
  • the injections may be made available in such forms as solutions, suspensions and emulsions, and as vehicles, sterilized water, water-propylene glycol, buffer solutions, and saline of 0.4 weight % concentration can be mentioned as examples.
  • These injections, in such liquid forms, may be frozen or lyophilized.
  • the latter products, obtained by lyophilization, are extemporaneously reconstituted with distilled water for injection or the like and administered.
  • the above forms of pharmaceutical composition can be prepared by formulating the compound having cortactin inhibitory action and a pharmaceutically acceptable carrier in the manner established in the art.
  • the pharmaceutically acceptable carrier includes various excipients, diluents, fillers, extenders, binders, disintegrators, wetting agents, lubricants, and dispersants, among others.
  • Other additives which are commonly used in the art can also be formulated. Depending on the form of pharmaceutical composition to be produced, such additives can be judiciously selected from among various stabilizers, fungicides, buffers, thickeners, pH control agents, emulsifiers, suspending agents, antiseptics, flavors, colors, tonicity control or isotonizing agents, chelating agents and surfactants, among others.
  • the pharmaceutical composition in any of such forms can be administered by a route suited to the objective disease, target organ, and other factors.
  • it may be administered intravenously, intraarterially, subcutaneously, intradermally, intramuscularly or via airways. It may also be directly administered topically into the affected tissue or even orally or rectally .
  • the dosage and dosing schedule of such a pharmaceutical preparation vary with the dosage form, the disease or its symptoms, and ' the patient's age and body weight, among other factors, and cannot be stated in general terms.
  • the usual dosage, in terms of the daily amount of the active ingredient for an adult human may range from about 0.0001 mg to about 500 mg, preferably about 0.001 mg. about. about 100 mg, and this amount can be administered once a day or in a few divided doses daily.
  • the composition may be provided in the form of a drug for gene therapy or a prophylactic drug.
  • the drug for gene therapy can be prepared by introducing the object polynucleotide into a vector or transfecting appropriate cells with the vector.
  • the modality of administration to a patient is roughly divided into two modes. The mode applicable to (1) the case in which a non- viral vector is used and the mode applicable to (2) the case in which a viral vector is used.
  • both the method of preparing a drug for gene therapy and the method of administration are dealt with in detail in several books relating to experimental protocols [e.g.
  • Bessatsu Jikken Igaku, Idenshi Chiryo-no-Kosogijutsu (Supplement to Experimental Medicine, Fundamental Techniques of Gene Therapy), Yodosha, 1996; Bessatsu Jikken Igaku: Idenshi Donyu & Hatsugen Kaiseki Jikken-ho (Supplement to Experimental Medicine: Experimental Protocols for Gene Transfer & Expression Analysis), Yodosha, 1997; Japanese Society for Gene Therapy (ed.): Idenshi Chiryo Kaihatsu Kenkyn Handbook (Research Handbook for Development of Gene Therapies), NTS, 1999, etc.].
  • any expression vector capable of expressing the anti- cortactin nucleic acid may be used. Suitable examples include pCAGGS (Gene 108: 193 (1991)), pBK-CMV, pcDNA 3.1, and pZeoSV (Invitrogen, Stratagene).
  • Transfer of a polynucleotide into the patient can be achieved by inserting the object polynucleotide into such a non-viral vector (expression vector) in the routine manner and administering the resulting recombinant expression vector.
  • a non-viral vector expression vector
  • the object polynucleotide can be introduced into the patient's cells or tissue.
  • the method of introducing the polynucleotide into cells includes the calcium phosphate transfection (coprecipitation) technique and the DNA (polynucleotide) direct injection method using a glass microtube, among others.
  • the method of introducing a polynucleotide into a tissue includes the polynucleotide transfer technique using internal type liposomes or electrostatic type liposomes, the
  • HVJ-liposome technique the modified HVJ-liposome (HVJ-AVE liposome) technique
  • receptor-mediated polynucleotide transfer technique the technique which comprises transferring the polynucleotide along with a vehicle (metal particles) into cells with a particle gun, the naked-DNA direct transfer technique, and the transfer technique using a positively charged polymer, among others.
  • Suitable viral vectors include vectors derived from recombinant adenoviruses and retrovirus. Examples include vectors derived from DNA or RNA viruses such as detoxicated retrovirus, adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, poxvirus, poliovirus, Sindbis virus, Sendai virus, SV40, human immunodeficiency virus (HIV) and so forth.
  • the adenovirus vector in particular, is known to be by far higher in infection efficiency than other viral vectors and, from this point of view, the adenovirus vector is preferably used.
  • Transfer of the polynucleotide into the patient can be achieved by introducing the object polynucleotide into such a viral vector and infecting the desired cells with the recombinant virus obtained. In this manner, the object polynucleotide can be introduced into the cells.
  • the method of administering the thus-prepared drug for gene therapy to the patient includes the in vivo technique for introducing the drug for gene therapy directly into the body and the ex vivo technique which comprises withdrawing certain cells from a human body, introducing the drug for gene therapy into the cells in vitro and returning the cells into the human body (Nikkei Science, April, 1994 issue, 20-45; Pharmaceuticals Monthly, 36(1), 23-48, 1994; Supplement to Experimental Medicine, 12(15), 1994; Japanese Society for Gene Therapy (ed.): Research Handbook for Development of Gene Therapies, NTS, 1991).
  • the drug is preferably introduced into the body by the in vivo technique.
  • the drug can be administered by a route suited to the object disease, target organ or the like.
  • it can be administered intravenously, intraarterially, subcutaneously or intramuscularly, for instance, or may be directly administered topically into the affected tissue.
  • the drug for gene therapy can be provided in a variety of pharmaceutical forms according to said routes of administration.
  • an injection can be prepared by the per se established procedure, for example by dissolving the active ingredient polynucleotide in a solvent, such as a buffer solution, e.g. PBS, physiological saline, or sterile water, followed by sterilizing through a filter where necessary, and filling the solution into sterile vitals, Where necessary, this injection may be supplemented with the ordinary carrier or the like.
  • a buffer solution e.g. PBS, physiological saline, or sterile water
  • the drug can be provided in various liposome- entrapped preparations in such forms as suspensions, frozen preparations and centrifugally concentrated frozen preparations.
  • a sustained-release preparation eg. a minipellet
  • the drug may be administered continuously and gradually to the affected site by means of an osmotic pump or the like.
  • the polynucleotide content of the drug for gene therapy can be judiciously adjusted according to the disease to be treated, the patient's age and body weight, and other factors but the usual dosage in terms of each polynucleotide is about 0.0001 to about 100 mg, preferably about 0.001 to about 10. mg. This amount is preferably administered several days or a few months apart.
  • the present invention also relates to methods for detecting the expression of cortactin polynucleotides or polypeptides in a sample (e.g., tissue or sample). Such methods can, for example, be utilized as part of prognostic and diagnostic evaluation of proliferative disorders and for the identification of subjects exhibiting predispositions to such disorders.
  • diagnosis and variants thereof, such as, but not limited to “diagnose”, “diagnosed” or “diagnosing” shall not be limited to a primary diagnosis of a clinical state, however should be taken to include any primary diagnosis or prognosis of a clinical state.
  • diagnosis assay formats described herein are equally relevant to monitoring inflammatory disease recurrence, or monitoring the efficiency of treatment of the disease. AU such uses of the assays described herein are encompassed by the present invention.
  • the present invention provides a method of diagnosing disorders associated with cell proliferation comprising the step of measuring the expression patterns of cortactin protein and/or mRNA. Yet another embodiment of a method of diagnosing disorders associated with cell proliferation comprising the step of detecting cortactin expression using anti-cortactin antibodies.
  • Such methods of diagnosis encompass the use of compositions, kits and other approaches for determining whether a patient is a candidate for treatment in which cortactin is targeted.
  • the present invention provides a method of diagnosing a proliferative disorder in a human or animal subject being tested, the method comprising contacting a biological sample from the subject being tested with a nucleic acid probe for a time and under conditions sufficient for hybridization to occur and then detecting the hybridization wherein a modified level of hybridization of the probe for the subject being tested compared to the hybridization obtained for a control subject not having a proliferative disorder indicates that the subject being tested has a proliferative disorder, and wherein the nucleic acid probe comprises a sequence selected from the group consisting of: (i) a sequence comprising at least about 20 contiguous nucleotides from SEQ ID NO:2 or 4;
  • modified level includes an enhanced, increased or elevated level of an integer being assayed, or alternatively, a reduced or decreased level of an integer being assayed.
  • a low stringency is defined herein as being a hybridization and/or a wash carried out in 6xSSC buffer, 0.1% (w/v) SDS at.28 0 C, or equivalent conditions.
  • a moderate stringency is defined herein as being a hybridization and/or washing carried out in 2xSSC buffer, 0.1% (w/v) SDS at a temperature in the range 45°C to 65°C, or equivalent conditions.
  • a high stringency is defined herein as being a hybridization and/or wash carried out in 0. IxSSC buffer, 0.1% (w/v) SDS, or lower salt concentration, and at a temperature of at least 65 0 C, or equivalent conditions. Reference herein to a particular level of stringency encompasses equivalent conditions using wash/hybridization solutions other than SSC known to those skilled in the art.
  • the stringency is increased by reducing the concentration of SSC buffer, and/or increasing the concentration of SDS and/or increasing the temperature of the hybridization and/or wash.
  • concentration of SSC buffer and/or increasing the concentration of SDS and/or increasing the temperature of the hybridization and/or wash.
  • the conditions for hybridization and/or wash may vary depending upon the nature of the hybridization matrix used to support the sample RNA, or the type of hybridization probe used.
  • an elevated, enhanced or increased level of expression of the nucleic acid is indicative of a proliferative disorder.
  • the hybridization comprises performing a nucleic acid hybridization reaction between a labeled probe and a second nucleic acid in the biological sample from the subject being tested, and detecting the label.
  • the hybridization comprises performing a nucleic acid amplification reaction eg., polymerase chain reaction (PCR), wherein the probe consists of a nucleic acid primer and nucleic acid copies of the nucleic acid in the biological sample are amplified.
  • PCR polymerase chain reaction
  • amplification may precede classical nucleic acid hybridization detection systems, to enhance specificity of detection, particularly in the case of less abundant mRNA species in the sample.
  • the polynucleotide is immobilised on a solid surface.
  • the present invention provides a method of diagnosing a proliferative disorder in a human or animal subject being tested, the method comprising contacting a biological sample from the subject being tested with an antibody for a time and under conditions sufficient for an antigen-antibody complex to form and then detecting the complex wherein a modified level of the antigen-antibody complex for the subject being tested compared to the amount of the antigen-antibody complex formed for a control subject not having a proliferative disorder indicates that the subject being tested has a proliferative disorder, and wherein the antibody binds specifically to a polypeptide having an amino acid sequence comprising at least about 10 contiguous amino acid residues of a sequence having at least about 80% identity to a sequence as shown in SEQ ID NO:1 or SEQ ID NO:3.
  • an elevated, enhanced or increased level of the antigen-antibody complex is indicative of a hyperproliferative disorder.
  • the present invention is not to be limited by the source or nature of the biological sample.
  • the biological sample is from a patient undergoing a therapeutic regimen to treat an a proliferative disorder.
  • the biological sample is from a patient suspected of having or developing a proliferative disorder.
  • the present invention provides a method of monitoring the efficacy of a therapeutic treatment of a disease associated with aberrant cell proliferation, the method comprising: (i) providing a biological sample from a patient undergoing the therapeutic treatment; and
  • the method further comprises comparing the level of the cortactin-associated transcript to a level of the cortactin-associated transcript in a biological sample from the patient prior to, or earlier in, the therapeutic treatment.
  • the present invention also provides a method of monitoring the efficacy of a therapeutic treatment of a disease associated with aberrant cell proliferation, the method comprising: (i) providing a biological sample from a patient undergoing the therapeutic treatment; and
  • the method further comprises comparing the level of the cortactin polypeptide to a level of the cortactin polypeptide in a biological sample from the patient prior to, or earlier in, the therapeutic treatment.
  • kits comprising polynucleotide probes and/or monoclonal antibodies, and optionally quantitative standards, for carrying out methods of the invention. Furthermore, the invention provides methods for evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of disorders as recited herein.
  • HNSCC human head and neck squamous cell carcinoma
  • Detroit 56, FaDu, Scc25 and Scc9 were maintained as previously described (Kalish et al, Clin. Cancer Res 10:7764-74 (2004)).
  • Retroviral infections using cortactin/pMIG were performed according to Brummer et al (J. Biol. Chem. 281, 626-637, 2006) and NIH3T3 culture and cell transformation assays were as described in Lynch and Daly (EMBO J, 21, 72-82, 2002).
  • EGF was from R&D Systems (Minneapolis, MN).
  • RNAL Suppression of cortactin expression by RNAL
  • 5'- GACUGGUUUUGGAGGCAAAUUUU-S ' SEQ ID NO:5
  • 5'-AAGCUGAGGGAGAAUGUCUUGU-S' SEQ ID NO:6
  • 5'-AAGACUGAGAAGCAUGCCUCTCC-S' SEQ ID NO:7
  • siRNA against lamin A/C was used as a control.
  • 20 ⁇ l of each single-stranded RNA 50 ⁇ M was incubated with 10 ⁇ l 5X annealing buffer (100 mM potassium acetate, 2 mM magnesium acetate, 30 mM HEPES at pH 7.4) for 1 min at 90°C and then 1 h at 37°C.
  • the RNA duplexes were then stored at -20 0 C until use.
  • Tubes 1 and 2 were incubated for 7-10 min at room temperature before combining the solutions. Following incubation for 20-25 min at room temperature, 152 ⁇ l of OptiMem was added to the mixture. This was then added to the cells, which were incubated with the siRNA for 2-4 days.
  • Cell Lysis, Immunoprecipitation and Immunoblotting were prepared using RIPA buffer (Lowenstein et al, Cell 70:431-42 (1992)) containing 10 ⁇ g/ml aprotinin, 10 ⁇ g/ml leupeptin, 1 mM sodium ortho vanadate, and 1 mM phenylmethylsulfonyl fluoride. Protein concentrations were determined using Protein Assay Reagent (Bio-Rad, Hercules, CA). Immunoprecipitations were performed by incubation with the indicated antibody for a minimum of 2 h at 4 0 C with constant mixing.
  • the cortactin monoclonal 4F11 and the Sprouty2 polyclonal antibody were purchased from Upstate Biotechnologies Inc. (Charlottesville, VA).
  • the monoclonal c-Cbl (for immunoprecipitations) and horseradish peroxidase-conjugated anti-phosphotyrosine RC20 antibodies were from BD Transduction Laboratories (Lexington, KY).
  • the polyclonal c-Cbl antibody R2 (for Western blotting) was a kind gift from Dr Wally Langdon (University of Western Australia).
  • Polyclonal EGFR 1005 was from Santa Cruz Biotechnology.
  • Antibodies against phospho- and total Erk and Akt were from Cell Signalling Technology.
  • the anti-beta actin antibody was from Sigma.
  • Example 1 Effect of altering cortactin expression levels on cell proliferation
  • HNSCC head and neck squamous cell carcinoma
  • Retroviral infection was used to generate stable pools of NIH-3T3 fibroblasts overexpressing cortactin (Figure 7A) which were then subjected to focus-forming assays.
  • Cells infected with the control retrovirus formed monolayers that were subject to contact inhibition and exhibited a low number of foci formed from spontaneously transformed cells.
  • the cortactin-overexpressing fibroblasts formed large numbers of transformed foci at confluence, indicating that cortactin exhibits a transforming potential in this system (Figure 7B).
  • Example 6 Effect of cortactin on colony formation by HNSCC cells under EGF-dependent conditions
  • Example 4 Since in Example 4 the colony formation assays were performed in the presence of 10% FCS, we next determined whether cortactin would enhance colony formation in the presence of a single mitogen. FaDu cells exhibiting siRNA-mediated knockdown of cortactin, or SCC9 cells transfected with a cortactin expression construct, were subjected to colony formation assays in the presence of 1% FCS and 10 ng/ml EGF. Addition of Iressa to these experiments confirmed that proliferation under these conditions was EGF-dependent. Again, in both cell models, high cortactin levels were associated with increased number and size of colonies ( Figure 8). Therefore, cortactin enhances cell proliferation in response to either serum or a single growth factor, specifically EGF.
  • cortactin siRNA RNA-binding protein

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Abstract

L’invention procure un procédé de criblage pour des composés qui modulent la prolifération cellulaire impliquant l’analyse de l’activité ou de l’expression de la cortactine en présence d’un composé candidat dans lequel l’activité modifiée ou l’expression de la cortactine lorsqu’on la compare à l’absence du composé indique un modulateur potentiel de la prolifération cellulaire. L’invention propose aussi des composés identifiés au moyen de ces procédés, des procédés de réduction ou d’amélioration de la prolifération cellulaire chez des sujets qui en ont besoin et des procédés pour le pronostic et l’évaluation diagnostique des maladies associées avec une prolifération cellulaire aberrante.
PCT/AU2006/000489 2005-04-11 2006-04-11 Procede de criblage pour des composes qui modulent la proliferation cellulaire WO2006108225A1 (fr)

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US9782381B2 (en) 2011-03-08 2017-10-10 University College Cardiff Consultants Limited Molecular targets for healing or treating wounds
CN107245502A (zh) * 2017-06-14 2017-10-13 中国科学院武汉病毒研究所 Cd2结合蛋白（cd2ap）和其相互作用蛋白
CN107245502B (zh) * 2017-06-14 2020-11-03 中国科学院武汉病毒研究所 Cd2结合蛋白(cd2ap)和其相互作用蛋白

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