WO2006058375A1

WO2006058375A1 - Identification of compounds for the treatment of proliferative disorders

Info

Publication number: WO2006058375A1
Application number: PCT/AU2005/001810
Authority: WO
Inventors: Peter William Gunning; Galina Schevzov; Justine Rachael Stehn
Original assignee: The Royal Alexandra Hospital For Children
Priority date: 2004-12-01
Filing date: 2005-12-01
Publication date: 2006-06-08

Abstract

The present invention relates to methods of screening for compounds that are useful in the treatment of proliferative disorders. The methods of screening comprise determining, in the presence of a candidate compound, the activity or cellular location of tropomyosin, the expression levels of tropomyosin, the nature of at least one expression product of one or more genes encoding tropomyosin, or the binding of tropomyosin to one of its binding partners.

Description

Identification of compounds for the treatment of proliferative disorders

Field of the Invention

Background of the Invention

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.

Cell proliferation involves the dynamic restructuring of the major proteins of the cytoskeleton, which are tubulin, actin and intermediate filament proteins. Each of these proteins can self-associate to form linear polymers of variable length, respectively referred to as microtubules, actin microfilaments, and intermediate filaments. During cell proliferation, the actin and tubulin cytoskeleton is restructured for accomplishing first mitotic spindle assembly, then chromosome segregation, and finally division of the cell into two daughters. Compounds which modulate the cytoskeleton's role in cell proliferation are therefore potentially useful in cancer chemotherapy.

As well as playing a fundamental role in cell proliferation, the cytoskeleton is also essential for intracellular transport, cell motility, and the establishment and regulation of cell polarity and organization. The ability of microfilaments to independently perform such a broad array of functions may be facilitated by the sorting of isoforms of the primary components of microfilaments to different intracellular compartments. Actin, for example, which provides the core microfilament polymer, is encoded by two isoforms in mammalian nonmuscle cells (Herman (1993) Curr. Opin. Cell Biol. 5:48- 55).

Tropomyosins (Tms), which are proteins associated with microfilaments, also exists in the form of numerous isoforms which are sorted to different intracellular components. The Tms are a group of proteins that bind by electrostatic charge to the helical groove of actin filaments and in striated muscle (together with troponins) regulate the interaction of the filaments with myosin in response to Ca²⁺. They are parallel, α- helical, coiled coil dimers which exist as both homo-dimers and hetero-dimers held together by a series of salt bridges and hydrophobic interactions. They may also interact through their N and C termini in solution (especially in low ionic strength buffers) and this can be monitored by viscosity. There are two major groups of Tms, the high and low molecular weight Tms. Muscle tissues primarily use high molecular weight Tms whereas non-muscle tissues can express both high and low molecular weight TMs at substantial levels.

The mammalian Tm gene family consists of four genes, BTΠI, BTm, TTm (Tm5 NM, where NM indicates nonmuscle), and ifTm (Tm4). Multiple isoforms (more than 40) of the Tm family are generated by alternative splicing (Helfman et al (1986) MoI. Cell. Biol. 6:3582-3595; Lees-Miller et al (1991) Bioessays 13:429-437), and the expression of these isoforms appears to be highly regulated. Many nonmuscle isoforms are generated from the ITm gene (Figure 1), and to date, 11 nonmuscle (NMl to NMIl) isoforms have been identified (Dufour et al (1998) J Biol. Chem. 273:18547-18555.). The exact role for all of the 11 nonmuscle isoforms is yet to be determined. Extensive spatial sorting of some Tm isoforms into different cellular compartments has been shown for several cell types (Percival et al (2000) Cell Motil Cytoskelet. 47:189-208; Weinberger et al (1996) J Neurosci. 16:238-252). Studies of Tm isoform sorting suggest that individual isoforms may confer specific functional properties to actin filaments. It has been reported that some higher-molecular- weight Tms (TmI, Tm2, and Tm3) are associated with stability of actin filaments and regulation of cell morphology and division (Boyd et al (1995) Proc. Natl. Acad. Sci. USA 92:11534- 11538; Gimona et al (1996) Proc. Natl. Acad. Sci. USA 93:9618-9623; Gunning et al (1997) Anat. Embryol. 195:311-315; Hughes et al (2003) GHa 42:25-35; Lin et al (1997) Int. Rev. Cytol. 170:1-38). More specifically, these reports indicate that high molecular- weight TMs are able to suppress tumorigenicity. In contrast, lower-molecular-weight Tm isoforms such as TTm nonmuscle (Tm5 NM) products have been reported at the leading edge of fibroblasts, suggesting that they may have a specific role in membrane organization, motility, and growth (Bryce et al (2003) MoI. Biol. Cell 14:1002-1016; Gunning et al (1998) Annu. Rev. Cell Dev. Biol. 14:339- 372; Lin et al (1988) J Cell Biol. 107:563-572; Lin et al (1997) Int. Rev. Cytol. 170:1- 38; Temm-Grove et al (1996) Cell Motil. Cytoskelet. 33:223-240). TTm gene products have been shown to be associated with Golgi apparatus-derived vesicles, which also suggests a role for low-molecular-weight Tms in vesicle trafficking (Heimann et al (1999) J Biol. Chem. 274:10743-10750).

Summary of the Invention

The present inventors have now found that specific Tm isoforms can regulate the stability of actin filament populations. In particular, Tm compositions can determine the sensitivity of specific actin filament populations to microfilament altering drugs. In other words, cells expressing a predominant tropomyosin isoform are likely to be more or less sensitive to certain drug treatments. Further, the present inventors have found that non-muscle Tm supply is limiting in vivo and regulates the amount of specific Tm/actin filaments.

These findings indicate for the first time that agents that selectively inhibit the activity or expression of specific TM isoforms are potential therapeutics for diseases associated with aberrant cell proliferation. In other words, specific Tm isoforms are ideal targets to use in screening for therapeutic agents that decrease the stability of actin filaments and thereby inhibit cell division or proliferation. Using specific Tm isoforms as targets for drug development enables disruption of specific filament populations rather than disruption of the entire cytoskeleton. It also enables inhibition of proliferation of specific cell types in cases where a specific Tm is enriched or uniquely expressed in a cancer cell.

Accordingly, the present invention provides a method of screening for an antiproliferative agent, the method comprising determining the activity or cellular location of one or more isoforms of cytoskeletal tropomyosin in the presence of a candidate compound, wherein alteration of the activity or cellular location of the one or more isoforms of cytoskeletal tropomyosin in the presence of the compound when compared to the absence of the compound indicates that the compound is a potential antiproliferative agent.

In the context of the present invention, it is preferred that the cytoskeletal tropomyosin isoform is a low molecular weight isoform. Non-limiting examples of suitable low molecular weight isoforms are Tm5a, Tm5b, TmBr-2, TmBr-3, Tm5NM-l, Tm5NM-2, Tm5NM-3, Tm5NM-4, Tm5NM-5, Tm5NM-6, Tm5NM-7, Tm5NM-8, Tm5NM-9, Tm5NM-10, Tm5NM-l l and Tm4.

In a preferred embodiment, the low molecular weight isoform is selected from the group consisting of Tm5a, Tm5b, Tm5NM-l, Tm5NM-2, Tm5NM-4, Tm5NM-5, Tm5NM-6, Tm5NM-7 and Tm4.

In a particularly preferred embodiment, the low molecular weight isoform is selected from the group consisting of Tm5NM-l, Tm5NM-2, Tm5NM-5 and Tm5NM-6. More preferably, the low molecular weight isoform is Tm5NM-l or Tm5NM-2.

In a preferred example, altered cellular location of an isoform of cytoskeletal tropomyosin, preferably loss of polarised distribution, in the presence of the compound indicates that the compound is a potential antiproliferative agent.

In one particular example, cellular location of the cytoskeletal tropomyosin isoform is assessed as an indicator of a potential antiproliferative agent. Cells which normally exhibit polarised distribution of tropomyosin (for example, gastrointestinal epithelial cells, fibroblasts or neurons) are preferably selected for this method of screening. Following exposure of the candidate compound to the selected cells, the location or distribution of a tropomyosin isoform is assessed and compared to the location or distribution of the same tropomyosin isoform in cells that have not been exposed to the candidate compound. In a preferred embodiment, loss of polarised distribution of the tropomyosin isoform in cells that have been exposed to the candidate compound indicates that the candidate compound is a potential antiproliferative agent.

In another example, this method of screening comprises determining the activity or cellular location of at least first and second isoforms of cytoskeletal tropomyosin in the presence of a candidate compound, wherein alteration of the activity or cellular location of the first isoform but not the second isoform in the presence of the compound when compared to the absence of the compound indicates that the compound is a potential antiproliferative agent.

Preferably, the first isoform is a low molecular weight isoform and the second isoform is a high molecular weight isoform. Preferably the high molecular weight isoform is selected from the group consisting of TmI₃ Tm2, Tm3 and Tm6.

The present invention also provides a method of screening for an antiproliferative agent, the method comprising determining the expression levels of one or more isoforms of cytoskeletal tropomyosin in the presence of a candidate compound, wherein altered expression of the one or more isoforms in the presence of the compound when compared to the absence of the compound indicates that the compound is a potential antiproliferative agent.

In a preferred example, reduced tropomyosin expression of the one or more cytoskeletal tropomyosin isoforms in the presence of the compound indicates that the compound is a potential antiproliferative agent.

In a preferred example, determining the expression level of the cytoskeletal tropomyosin isoform comprises measuring the amount of the tropomyosin protein and/or mRNA. In one preferred embodiment, the amount of tropomyosin protein is measured using an anti-tropomyosin antibody. In another embodiment, the amount of the tropomyosin-associated transcript (e.g. mRNA) is measured by contacting the sample with a polynucleotide that selectively hybridizes to the tropomyosin transcript.

In another example, this method of screening comprises determining the expression levels of at least first and second isoforms of cytoskeletal tropomyosin in the presence of a candidate compound, wherein alteration of the expression levels of the first isoform but not the second isoform in the presence of the compound when compared to the absence of the compound indicates that the compound is a potential antiproliferative agent.

Preferably, the first isoform is a low molecular weight isoform and the second isoform is a high molecular weight isoform. Preferably the high molecular weight isoform is selected from the group consisting of TmI, Tm2, Tm3 and Tm6. The present invention also provides a method of screening for an antiproliferative agent, the method comprising determining the nature of at least one expression product of one or more genes encoding cytoskeletal tropomyosin in the presence of a 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 antiproliferative agent.

The expression product analysed in this method may be, for example, mRNA or a protein. In one preferred example, the expression product is mRNA and the method involves detecting alternative spliced forms of an mRNA transcript when expression of the gene occurs in the presence of the candidate compound. In another example, the expression product is a 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.

In another example, this method of screening comprises determining the nature of at least a first and second expression product of one or more genes encoding cytoskeletal tropomyosin in the presence of a candidate compound, wherein alteration of the nature of the first expression product but not the second expression product in the presence of the compound when compared to the absence of the compound indicates that the compound is a potential antiproliferative agent.

Preferably, the first expression product encodes or consists of a low molecular weight isoform of cytoskeletal tropomyosin and the second expression product encodes or consists of a high molecular weight isoform of cytoskeletal tropomyosin. Preferably the high molecular weight isoform is selected from the group consisting of TmI₃ Tm2, Tm3 and Tm6.

In yet a further aspect the present invention provides a method of screening for an antiproliferative agent, the method comprising measuring the binding of one or more isoforms of cytoskeletal tropomyosin to one of its binding partners in the presence of a candidate compound, wherein an altered level of binding of the one or more isoforms to its binding partner in the presence of the compound when compared to the absence of the compound indicates that the compound is a potential antiproliferative agent. In a preferred example a reduced level of binding of the isoform of tropomyosin to its binding partner in the presence of the compound indicates that the compound is a potential antiproliferative agent.

In a further preferred example the tropomyosin binding partner is selected from the group consisting of calponin, CEACAMl₃ endostatin, Enigma, Gelsolin (preferably sub-domain 2), S100A2 and actin. In a further preferred example, the tropomyosin binding partner is actin.

In another example, this method of screening comprises determining the level of binding of at least first and second isoforms of cytoskeletal tropomyosin to their binding partners in the presence of a candidate compound, wherein alteration of the level of binding of the first isoform but not the the second isoform in the presence of the compound when compared to the absence of the compound indicates that the compound is a potential antiproliferative agent.

Preferably, the first isoform is a low molecular weight isoform and the second isoform is a high molecular weight isoform. Preferably the high molecular weight isoform is selected from the group consisting of TmI, Tm2, Tm3 and Tm6.

As will be readily understood by those skilled in this field the methods of the present invention provide a rational method for designing and selecting compounds which interact with and modulate the activity of tropomyosin. 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 include such further developmental steps. For example, it is intended that embodiments of the present invention further include manufacturing steps such as selecting or purifying the identified candidate compound and/or incorporating the compound into a pharmaceutical composition in the manufacture of a medicament for the treatment of a proliferative disease.

By "proliferative disease" is meant a disease that is caused by or results in inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. For example, cancers such as lymphoma, leukemia, melanoma, ovarian cancer, breast cancer, pancreatic cancer, bladder cancer, gastric cancer, salivary gland carcinoma, and lung cancer are all examples of proliferative disease. A myeloproliferative disease is another example of a proliferative disease.

Accordingly, in preferred examples of the present invention the method of screening further comprises formulating the identified compound for administration to a human or a non-human animal as described herein.

The present invention also provides a method of screening for an antiproliferative agent, the method comprising (a) determining the activity or cellular location of one or more isoforms of cytoskeletal tropomyosin in the presence of a candidate compound, wherein alteration of the activity or cellular location of the one or more isoforms in the presence of the compound when compared to the absence of the compound indicates that the compound is a potential antiproliferative agent; (b) optionally, determining the structure of the candidate compound; and

(c) providing the candidate compound or the name or structure of the candidate compound.

The present invention also provides a method of screening for an antiproliferative agent, the method comprising

(a) determining the expression levels of one or more isoforms of cytoskeletal tropomyosin in the presence of a candidate compound, wherein altered expression levels of the one or more isoforms in the presence of the compound when compared to the absence of the compound indicates that the compound is a potential antiproliferative agent;

(b) optionally, determining the structure of the candidate compound; and

(a) determining the nature of at least one expression product of one or more genes encoding cytoskeletal tropomyosin in the presence of a 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 antiproliferative agent; (b) optionally, determining the structure of the candidate compound; and

(a) measuring the binding of one or more isoforms of cytoskeletal tropomyosin to one of its binding partners in the presence of a candidate compound, wherein an altered level of binding of the one or more isoforms to its binding partner in the presence of the compound when compared to the absence of the compound indicates that the compound is a potential antiproliferative agent;

(b) optionally, determining the structure of the candidate compound; and

Naturally, for agents that are known albeit not previously tested for their function using a screen provided by the present invention, determination of the structure of the compound is implicit in step (b). 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.

As used herein, the term "providing the agent" shall be taken to include any chemical or recombinant synthetic means for producing said agent or alternatively, the provision of an agent that has been previously synthesized by any person or means.

For example, a peptidyl compound is synthesized using is produced synthetically. Synthetic peptides are prepared using known techniques of solid phase, liquid phase, or peptide condensation, or any combination thereof, and can include natural and/or unnatural amino acids. Amino acids used for peptide synthesis may be standard Boc (Nα-amino protected Nα-t-butyloxycarbonyl) amino acid resin with the deprotecting, neutralization, coupling and wash protocols of the original solid phase procedure of Merrifield, J Am. Chem. Soc, 55:2149-2154, 1963, or the base-labile Nα-amino protected 9-fluorenylmethoxycarbonyl (Fmoc) amino acids described by Carpino and Han, J Org. Chem., 57:3403-3409, 1972. Both Fmoc and Boc Nα-amino protected amino acids can be obtained from various commercial sources, such as, for example, Fluka, Bachem, Advanced Chemtech, Sigma, Cambridge Research Biochemical, Bachem, or Peninsula Labs. Synthetic peptides are alternatively produced using techniques known in the art and described, for example, in Stewart and Young (In: Solid Phase Synthesis, Second

Edition, Pierce Chemical Co., Rockford, 111. (1984) and/or Fields and Noble (Int. J. Pept. Protein Res., 35:161-214, 1990), or using automated synthesizers. Accordingly, peptides of the invention may comprise D-amino acids, a combination of D- and L- amino acids, and various unnatural amino acids (e.g., β-methyl amino acids, Cα-methyl amino acids, and Nα-methyl amino acids, etc) to convey special properties. Synthetic amino acids include ornithine for lysine, fmorophenylalanine for phenylalanine, and norleucine for leucine or isoleucine.

In another embodiment, a peptidyl agent is produced using recombinant means. For example, an oligonucleotide or other nucleic acid (eg., a nucleic acid encoding a dominant negative inhibitor of the protein of interest) is placed in operable connection with a promoter. Methods for producing such expression constructs, introducing an expression construct into a cell and expressing and/or purifying the expressed peptide, polypeptide or protein are known in the art.

Alternatively, the peptide, polypeptide or protein is expressed using a cell free system, such as, for example, the TNT system available from Promega. Such an in vitro translation system is useful for screening a peptide library by, for example, ribosome display, covalent display or mRNA display.

Methods for producing antibodies, preferably a monoclonal antibody, or a fragment or recombinant fragment thereof are described herein.

In a preferred embodiment, the compound 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 method for the treatment or prevention of a proliferative disease, the method comprising administering to the cell an agent that modulates expression, location or activity of one or more isoforms of cytoskeletal tropomyosin. In the context of the present invention, the candidate compound or agent may be selected from the group consisting of a peptide, an antibody directed against tropomyosin, a small organic molecule, a sugar, a polysaccharide, an antisense compound directed against tropomyosin-encoding niRNA, an anti-tropomyosin catalytic molecule such as a ribozyme or a DNAzyme, and a dsRNA or small interfering RNA (RNAi) molecule that targets tropomyosin expression.

In one preferred embodiment the agent is a tropomyosin antagonist. Preferably, the tropomyosin antagonist is an antisense compound, a catalytic molecule or an RNAi molecule directed against tropomyosin-encoding mRNA. In a further preferred embodiment, the tropomyosin antagonist is an antisense compound, a catalytic molecule or an RNAi molecule targeted specifically against exon Ib or 9d of the TPM 1 gene (SEQ ID NO:7) or exon Ib or 9d of the TPM 3 gene (SEQ ID NO:8).

In a further preferred embodiment the tropomyosin antagonist is an antisense compound, a catalytic molecule or an RNAi molecule targeted to the sequence AGCTCGCTGGAGGCGGTG (SEQ ID NO: 13).

In one particularly preferred embodiment, the tropomyosin antagonist is an antisense compound comprising the sequence CACCGCCUCCAGCGAGCT (SEQ ID NO: 14).

In a preferred embodiment the tropomyosin antagonist specifically alters the cellular location of Tm5a, Tm5b, Tm5NM-l, Tm5NM-2, Tm5NM-4, Tm5NM-5, Tm5NM-6, Tm5NM-7 or Tm4. By "specifically alters the cellular location of Tm5a, Tm5b, Tm5NM-l, Tm5NM-2, Tm5NM-4, Tm5NM-5, Tm5NM-6, Tm5NM-7 or Tm4" we mean that the compound significantly alters the cellular location of Tm5a, Tm5b, Tm5NM-l, Tm5NM-2, Tm5NM-4, Tm5NM-5, Tm5NM-6, Tm5NM-7 or Tm4 without significantly altering the cellular location of other tropomyosin isoforms.

In another preferred embodiment the tropomyosin antagonist specifically reduces or inhibits Tm5a, Tm5b, Tm5NM-l, Tm5NM-2, Tm5NM-4, Tm5NM-5, Tm5NM-6, Tm5NM-7 or Tm4 expression. By "specifically reduces or inhibits Tm5a, Tm5b, Tm5NM-l, Tm5NM-2, Tm5NM-4, Tm5NM-5, Tm5NM-6, Tm5NM-7 or Tm4 expression" we mean that the compound significantly reduces or inhibits Tm5a, Tm5b, Tm5NM-l, Tm5NM-2, Tm5NM-4, Tm5NM-5, Tm5NM-6, Tm5NM-7 or Tm4 expression without significantly reducing or inhibiting the expression of other tropomyosin isoforms.

In another preferred embodiment the tropomyosin antagonist specifically alters the binding of Tm5a, Tm5b, Tm5NM-l, Tm5NM-2, Tm5NM-4, Tm5NM-5, Tm5NM-6,

Tm5NM-7 or Tm4 to one of its binding partners. By "specifically alters the binding of

Tm5a, Tm5b, Tm5NM-l, Tm5NM-2, Tm5NM-4, Tm5NM-5, Tm5NM-6, Tm5NM-7 or Tm4 to one of its binding partners" we mean that the compound significantly alters the binding of Tm5a, Tm5b, Tm5NM-l, Tm5NM-2, Tm5NM-4, Tm5NM-5, Tm5NM- 6, Tm5NM-7 or Tm4 to one of its binding partners without significantly altering the binding of other tropomyosin isoforms to their binding partners.

The present invention also provides 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.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

The various features and embodiments of the present invention, referred to in individual sections above apply, as appropriate, to other sections. Consequently features specified in one section may be combined with features specified in other sections, as appropriate.

Brief Description of the Figures

Figure 1. Tropomyosin and actin gene maps and protein products. Mammalian Tm gene structures and their protein products. α-Tm gene (A) and protein products (B). β-Tm gene (C) and protein products (D). γ-Tm gene (E) and protein products (F). δ-Tm gene (G) and protein products (H) (Adapted from Dufour et al, 1998 and Lees-miller and Helfman, 1991). N-terminal protein sequence for mammalian non-muscle and muscle actin isoforms (I) (adapted from Herman 1993). Figure 2. Non-muscle Tm levels are limiting.

(A) Whole brain tissue was extracted from WT₃ transgenic Tm5 (5/52) and transgenic Tm3 (3/66) animals. lOμg of brain tissue lysate was separated using 15%SDS PAGE and, the western blots are shown. Tm3 expression was determined using the WSα/9d antibody (which also recognises Tm6, Tm5a and TmI). Tm5NMl expression could be detected using a variety of antibodies. The overexpressed human Tm5NMl expression was determined using the human specific LCl antibody. All Tm5NM products from the α-Tm gene, including Tm5NMl can be detected using the CG3 antibody. γ9d recognises only a subset of Tm5NM products that contain the 9d exon, specifically Tm5NMl and Tm5NM2. (B) lOug of lysates from wild type and 9d+/- embryonic stem cells, brain, kidney, lung and stomach were analysed as described in A. The products containing the 9d exon, Tm5NMl and Tm5NM2, were detected using the gamma/9d antibody and migrating with an apparent molecular weight of 30kd. Note that the higher molecular weight bands detected at about 38kd in stomach are not products of the gamma Tm gene.

Figure 3. Changes in Tm expression alter the subcellular distribution ofnon- muscle actins.

(A) Triton-soluble and —insoluble lysates prepared from B35 rat neuronal cells over expressing Tm3, hTm5NMl or vector alone were separated using 15% SDS-PAGE.

Subcellular localisation of the and β-and γ-actins were detected using specific antibodies. Tm3 was detected using the 311 antibody (which also recognises TmI, Tm2 and Tm6) and hTm5NMl was detected using the human specific LCl antibody.

(B) Densitometry of n=3 western blots was analysed and the ratio of Tm or actin found in the soluble versus the insoluble fraction for all three cell types is presented in the graphs.

Figure 4. EYFP-TmSNMl and ECFP -Tm3 fusion constructs.

(A) hTm5NMl with the 3¹ UTR was ligated in frame to the carboxyl terminus of EYFP in the pEYFP-Cl expression vector. (B) hTm3 was ligated in frame to the carboxyl terminus of ECFP in the pECFP-Cl expression vector.

Figure 5. 9d knock-out targeting construct Targeting construct used to generate exon 9d knock-out mice. Key to Sequence Listing;

SEQ ID N0:l: Homo sapiens cDNA sequence for an isoform encoded by the tropomyosin 1 (alpha) (TPM 1) gene sequence; SEQ ID NO:2: Homo sapiens cDNA sequence for an isoform encoded by the tropomyosin 2 (beta) (TPM 2) gene sequence;

SEQ ID NO:3: Homo sapiens cDNA sequence for an isoform encoded by the tropomyosin 3 (TPM 3) gene sequence;

SEQ ID NO:4: Homo sapiens cDNA sequence for an isoform encoded by the tropomyosin 4 (TPM 4) gene sequence;

SEQ ID NO:5: Homo sapiens cDNA sequence of isoform TM5a;

SEQ ID NO:6: Homo sapiens cDNA sequence of isoform TM5b;

SEQ ID NO:7: Homo sapiens DNA sequence of exon Ib of the TPMl gene;

SEQ ID NO: 8: Homo sapiens DNA sequence of exon Ib of the TPM3 gene; SEQ ID NO:9: Homo sapiens protein sequence of isoform TM5a;

SEQ ID NO: 10: Homo sapiens protein sequence of isoform TM5b;

SEQ ID NO:11: Homo sapiens protein sequence of exon lb ofthe TPMl gene;

SEQ ID NO:12: Homo sapiens protein sequence of exon Ib of the TPM3 gene;

SEQ ID NO: 13: Homo sapiens target sequence within exon Ib of the TPMl gene for preferred antisense constructs;

SEQ ID NO: 14: Antisense oligonucleotide sequence targeted to exon Ib of the TPMl gene;

SEQ ID NO: 15: Nonsense oligonucleotide sequence (control sequence);

SEQ ID NOs: 16 and 17: Polynucleotides for producing siRNA molecules which downregulate human TM5a or TM5b production;

SEQ ID NOs: 18-20 - Antigenic epitopes in the amino acid sequence encoded by exon

Ib of the TMPl gene.

Detailed Description of the Preferred Embodiments

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g. in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods (see generally, Sambrook et aL, Molecular Cloning: A Laboratory Manual, 3rd ed. (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. and Ausubel et al., Short Protocols in Molecular Biology (1999) 4th Ed, John Wiley & Sons, Inc. - and the full version entitled Current Protocols in Molecular Biology, which are incorporated herein by reference) and chemical methods.

Tropomyosins

Tropomyosins (TMs) are a diverse group of proteins found in all eukaryotic cells, with distinct isoforms found in muscle (skeletal, cardiac and smooth), brain and various non- muscle cells. They are elongated proteins that possess a simple dimeric I-helical coiled coil structure along their entire length. The coiled coil structure is based on a repeated pattern of seven amino acids with hydrophobic residues at the first and fourth positions and is highly conserved in all TM isoforms found in eukaryotic organisms from yeast to man with a prominent seven-residue periodicity (five motifs). Different isoforms are produced by differential splicing; e.g isoforms of I-tropomyosin differ in striated and smooth muscle.

TMs are associated with the thin filaments in the sarcomeres of muscle cells and the microfilaments of non-muscle cells. The TMs bind to themselves in a head-to-tail manner, and lie in the groove of F-actin, with each molecule interacting with six or seven actin monomers.

The function of TM in skeletal and cardiac muscle is, in association with the troponin complex (troponins T, C and I), to regulate the calcium-sensitive interaction of actin and myosin. Under resting intracellular calcium ion concentrations, the troponin- tropomyosin complex inhibits actomyosin ATPase activity. When a stimulus induces calcium ion release in the muscle cell, troponin-C binds additional calcium ions and a conformational change is transmitted through the troponin-tropomyosin complex which releases the inhibition of actomyosin ATPase activity, resulting in contraction.

In contrast to the skeletal and cardiac muscle, the biological functions of smooth muscle and non-muscle TMs are poorly understood. Smooth muscle and non-muscle cells are devoid of a troponin complex and the phosphorylation of the light chains of myosins appears to be the major calcium-sensitive regulatory mechanism controlling the interaction of actin and myosin. These differences in the regulation of contractile apparatus of various cell types appear to require structurally as well as functionally distinct forms of TM. When used herein the term "tropomyosin" is intended to encompass all isoforms of the protein. For example, the term encompasses all isoforms encoded by the mammalian genes TPM 1 (also known as the alpha-TM gene) (MacLeod and Gooding, 1988, MoL Cell. Biol. 8, 433-440), TPM 2 (also known as the beta-TM gene) (MacLeod et al, 1985, Proc. Natl. Acad. Sci. USA 82, 7835-7839), TPM 3 (also known as the gamma- TM gene) (Clayton et al, 1988, J. MoI. Biol. 201, 507-515), and TPM 4 (also known as the delta-TM gene) (MacLeod et al, 1987, J. MoI. Biol. 194, 1-10).

There are at least 40 tropomyosin isoforms that are derived from these four genes by alternative splicing (Figure 1). See, for example, Lees-Miller and Helfman, 1991, Bioessays 13(9):429-437. Although tropomyosin isoforms have a high degree of similarity, there are some differences in the actin binding and head-tail binding domains. The various tropomyosin isoforms have different binding affinities for actin. In addition, the tropomyosin position on the actin microfilament may modulate actin' s role in cell motility and cytoskeletal remodelling. Once inserted, tropomyosins influence the interaction between actin and other actin binding proteins. For example, high molecular weight tropomyosins are protective against the severing activity of the actin binding protein gelsolin.

When used herein the term "cytoskeletal tropomyosin" encompasses all of the tropomyosin isoforms depicted in Figure 1 except a-Υmfast, a-Tmslow and β-Tm.

cDNA sequences of isoforms encoded by the human TPMl, TMP2, TPM3 and TPM4 genes are shown in SEQ ID NOs 1 to 4 respectively. These sequences are representative examples only and are not intended to limit the scope of the present invention. The methods of the present invention may be targeted to other human or non-human tropomyosin sequences.

Tropomyosin antagonists/agonists

In one aspect the present invention relates to methods of screening for compounds that regulate tropomyosin activity or location within a cell.

In certain embodiments, combinatorial libraries of potential modulators will be screened for an ability to bind to a tropomyosin or to modulate activity. Conventionally, new chemical entities with useful properties are generated by identifying a chemical compound (called a "lead compound") with some desirable property or activity, e.g., inhibiting activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds. Often, high throughput screening (HTS) methods are employed for such an analysis.

In one preferred embodiment, high throughput screening methods involve providing a library containing a large number of potential therapeutic compounds (candidate compounds). Such "combinatorial chemical libraries" are then screened in one or more assays to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical "building blocks" such as reagents. For example, a linear combinatorial chemical library, such as a polypeptide (e.g., mutein) library, is formed by combining a set of chemical building blocks called amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks (Gallop et al, 1994, J Med. Chem. 37(9):1233-1251).

Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries, peptoids, encoded peptides, random bio-oligomers, nonpeptidal peptidomimetics, analogous organic syntheses of small compound libraries, nucleic acid libraries, peptide nucleic acid libraries, antibody libraries, carbohydrate libraries and small organic molecule libraries.

Tropomyosin binding compounds can be readily identified and isolated by methods known to those of skill in the art. Examples of methods that may be used to identify tropomyosin binding compounds are the yeast-2-hybrid screening, phage display, affinity chromatography, expression cloning, Biacore systems, immunoprecipitation and GST pull downs coupled with mass spectroscopy. Biacore systems are used to identify chemical mimetics of a tropomyosin protein as these systems enable direct detection and monitoring of biomolecular binding events in real time without labeling and often without purification of the substances involved. (Biacore, Rapsagatan 7, SE 754 50 Uppsala.).

In particular, the yeast-2-hybrid screening approach utilizes transcription activation to detect protein-protein interactions. Many transcription factors can be separated into two domains, a DNA binding domain and a transcriptional activation domain that are inactive when separated. When the two domains are brought into "close proximity' their functional transcriptional activation activity is recreated. In the present invention, a tropomyosin protein is fused to a transcription factor DNA binding domain and cDNAs from a cDNA library are fused to a sequence encoding a transcriptional activation domain. A yeast strain which has been transformed with the cDNA encoding the protein of interest fused to a transcription factor DNA binding domain, is transformed with the transcriptional activation domain/cDNA fusion library. Any cDNA which codes a protein that binds to the protein of interest will allow the formation of a functional hybrid transcriptional activator (as the DNA binding and transcriptional activation domains are now in 'close proximity') leading to the expression of a reporter gene that results in cell survival. The cDNA coding the binding protein is then isolated and the protein that it encodes identified.

The assays to identify modulators are preferably amenable to high throughput screening. Preferred assays thus detect enhancement or inhibition of tropomyosin gene transcription, inhibition or enhancement of polypeptide expression, and inhibition or enhancement of polypeptide activity.

High throughput assays for the presence, absence, quantification, or other properties of particular nucleic acids or protein products are well known to those of skill in the art, as are high throughput assays for redistribution of GFP-tagged proteins. Similarly, binding assays and reporter gene assays are similarly well known. Thus, e.g., U.S. Patent No. 5,559,410 discloses high throughput screening methods for proteins, U.S. Patent No. 5,585,639 discloses high throughput screening methods for nucleic acid binding (i.e., in arrays), while U.S. Patent Nos. 5,576,220 and 5,541,061 disclose high throughput methods of screening for ligand/antibody binding. In addition, high throughput screening systems are commercially available (see, e.g., Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman Instruments, Inc. Fullerton, CA; Precision Systems, Inc., Natick, MA, etc.). These systems typically automate entire procedures, including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detectors) appropriate for the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols for various high throughput systems. Thus, e.g., Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding, and the like.

Protein or Peptide inhibitors

In one embodiment, candidate compounds are proteins, which may be naturally occurring proteins or fragments of naturally occurring proteins. Thus, e.g., cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts, may be used. In this way libraries of proteins may be made for screening in the methods of the invention. 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. Particularly useful test compound will be directed to the class of proteins to which the target belongs, e.g., substrates for enzymes or ligands and receptors.

In a preferred embodiment, 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 as is outlined above, random peptides, or "biased" random peptides. By "randomized" or grammatical equivalents herein is meant that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. Since generally these random peptides (or nucleic acids, discussed below) are chemically synthesized, they may incorporate any nucleotide or amino acid at any position. The synthetic process can be designed to generate randomized proteins or nucleic acids, 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 agents. In one embodiment, peptidyl tropomyosin inhibitors are chemically or recombinantly synthesized as oligopeptides (approximately 10-25 amino acids in length) derived from a tropomyosin sequence. Alternatively, tropomyosin fragments are produced by digestion of native or recombinantly produced tropomyosin by, for example, using a protease, e.g., trypsin, thermolysin, chymotrypsin, or pepsin. Computer analysis (using commercially available software, e.g. MacVector, Omega, PCGene, Molecular Simulation, Inc.) is used to identify proteolytic cleavage sites. The proteolytic or synthetic fragments can comprise as many amino acid residues as are necessary to partially or completely inhibit tropomyosin 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.

Protein or peptide inhibitors may also be dominant-negative mutants of tropomyosin. The term "dominant-negative mutant" refers to a tropomyosin polypeptide that has been mutated from its natural state and that interacts with a protein that tropomyosin normally interacts with thereby preventing endogenous native tropomyosin from forming the interaction.

Anti-tropomyosin Antibodies

The term "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 tropomyosin. These antibody fragments retain some ability to selectively bind with its antigen and are defined as follows:

(1) 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;

(2) Fab¹, 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 Fab¹ fragments are obtained per antibody molecule; (3) (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 Fab¹ fragments held together by two disulfide bonds;

(4) 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; and

(5) Single chain antibody ("SCA"), 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.

Methods of making these fragments are known in the art. (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988), incorporated herein by reference).

Antibodies of the present invention can be prepared using intact tropomyosin 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. Such commonly used carriers which are chemically coupled to the peptide include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid. The coupled peptide may then be used to immunize the animal (e.g., a mouse or a rabbit).

If desired, 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. 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 al. Nature 256, 495-497, 1975; Kozbor et ah, J. Immunol. Methods 81, 31-42, 1985; Cote et ah, Proc. Natl. Acad. Sci. USA 80, 2026- 2030, 1983; Cole et al., MoI. Cell Biol. 62, 109-120, 1984).

Methods known in the art allow antibodies exhibiting binding for tropomyosin to be identified and isolated from antibody expression libraries. For example, a method for the identification and isolation of an antibody binding domain which exhibits binding to tropomyosin is the 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 al., Proc. Nat. Acad. Sci., 87:8095-8099, 1990). This methodology can also be applied to hybridoma cell lines expressing monoclonal antibodies with binding for a preselected ligand. 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 al., Plenum Press, 1980; and U.S. 4,172,124, incorporated by reference.

In addition, methods of producing chimeric antibody molecules with various combinations of "humanized" antibodies are known in the art and include combining murine variable regions with human constant regions (Cabily, et al. Proc. Natl. Acad. Sci. USA, 81:3273, 1984), or by grafting the murine-antibody complementarity determining regions (CDRs) onto the human framework (Riechmann, et al., Nature 332:323, 1988).

In one embodiment, the antibody binds at least a portion of a region of human tropomyosin selected from, but not limited to, the group consisting of SEQ ID NOs: 18 - 20.

Antisense compounds

The term "antisense compounds" encompasses DNA or RNA molecules that are complementary to at least a portion of a tropomyosin mRNA molecule (Izant and Weintraub, Cell 36:1007-15, 1984; Izant and Weintraub, Science 229(4711):345-52,

1985) and capable of interfering with a post-transcriptional event such as mRNA translation. Antisense oligomers complementary to at least about 15 contiguous nucleotides of tropomyosin-encoding mRNA are preferred, since they are easily synthesized and are less likely to cause problems than larger molecules when introduced into the target tropomyosin-producing cell. The use of antisense methods is well known in the art (Marcus- S akura, Anal. Biochem. 172: 289, 1988). Preferred antisense nucleic acid will comprise a nucleotide sequence that is complementary to at least 15 contiguous nucleotides of a sequence encoding the amino acid sequence set forth in SEQ ID NO:7 or SEQ ID NO:8.

Catalytic nucleic acids

The term 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, C, G, T and U, as well as derivatives thereof. Derivatives of these bases are well known in the art.

Typically, 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"). To achieve specificity, preferred ribozymes and DNAzymes will comprise a nucleotide sequence that is complementary to at least about 12-15 contiguous nucleotides of a sequence encoding a tropomyosin isoform.

The types of ribozymes that are particularly useful in this invention are the hammerhead ribozyme (Haseloff and Gerlach 1988, Perriman et ah, 1992) and the hairpin ribozyme (Shippy et ah, 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. Accordingly, also provided by this invention is a nucleic acid molecule, i.e., DNA or cDNA, coding for the ribozymes of this invention. When the vector also contains an RNA polymerase promoter operably linked to the DNA molecule, the ribozyme can be produced in vitro upon incubation with RNA polymerase and nucleotides. In a separate embodiment, the DNA can be inserted into an expression cassette or transcription cassette. After synthesis, 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. Alternatively, 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.

RNA inhibitors

dsRNA is particularly useful for specifically inhibiting the production of a particular protein. Although not wishing to be limited by theory, Dougherty and Parks (Curr. Opin. Cell Biol. 7: 399 (1995)) 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 a tropomyosin protein. Conveniently, 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 tropomyosin 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.

As used herein, the terms "small interfering RNA" (siRNA), and "RNAi" refer to homologous double stranded RNA (dsRNA) that specifically targets a gene product, thereby resulting in a null or hypomorphic phenotype. Specifically, the dsRNA comprises two short nucleotide sequences derived from the target RNA encoding PAC- 1 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 al., Nature 391, 806-811, 1998, and reviewed by Sharp, Genes & Development, 13, 139-141, 1999). Preferred siRNA molecules comprise a nucleotide sequence that is identical to about 19-21 contiguous nucleotides of the target mRNA. Preferably, the target sequence is exon Ib of the TPMl or TMP3 genes.

As exemplified herein, preferred siRNA against a tropomyosin encoding region comprises a 21-nucleotide sequence set forth in SEQ ID NO:16 or SEQ ID NO: 17. For producing siRNA which include a stem loop structure from the exemplified siRNAs set forth in SEQ ID NOS: 16 and 17, the sense and antisense strands are positioned such that they flank an intervening loop sequence. Preferred loop sequences will be known to those skilled in the art.

Small molecule inhibitors

Numerous organic molecules may be assayed for their ability to modulate the immune system. For example, within one embodiment of the invention 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 deconvoluted to determine and isolate the most active compounds.

Representative examples of such 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. et al, "Preparation of library of resin-bound aromatic carbocyclic compounds," WO 95/16712; Ellman, J. A., "Solid phase and combinatorial synthesis of benzodiazepine compounds on a solid support," U.S. Pat. No. 5,288.514; Felder, E. et al, "Novel combinatorial compound libraries," WO 95/16209: Lerner. R. et al, "Encoded combinatorial chemical libraries." WO 93/20242; Pavia, M. R. et al, "A method for preparing and selecting pharmaceutically useful non-peptide compounds from a structurally diverse universal library," WO 95/04277; Summerton, J. E. and D. D. Weller, "Morpholino-subunit combinatorial library and method," U.S. Pat. No. 5,506,337; Holmes, C, "Methods for the Solid Phase Synthesis of Thiazolidinones, Metathiazanones, and Derivatives thereof," WO 96/00148; Phillips, G. B. and G. P. Wei, "Solid-phase Synthesis of Benzimidazoles," Tet. Letters 37:4887-90, 1996; Ruhland, B. et al, "Solid-supported Combinatorial Synthesis of Structurally Diverse .beta.-Lactams," J. Amer. Chem. Soc. 111 :253-4, 1996; Look, G. C. et al, "The Identification of Cyclooxygenase-1 Inhibitors from 4-Thiazolidinone Combinatorial Libraries," Bioorg and Med. Chem. Letters 6:707-12, 1996.

Candidate compounds may be organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons. Preferred small molecules are less than 2000, or less than 1500 or less than 1000 or less than 500 Daltons. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

In one embodiment, 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.

In one embodiment of the present invention, the candidate compound is 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. In one particular embodiment, the candidate compound is obtained from an extract of a strain of Enteropathogenic E. coli (EPEC). Methods of screening for tropomyosin agonists/antagonists

Screening protocol based on polarised distribution of tropomyosin

An example of a screening method in which the ability of a candidate compound to inhibit tropomyosin function may involve an analysis of the effect of the compound on polarised distribution of tropomyosin within a cell.

For example, cells expressing a labelled tropomyosin isoform of interest may be exposed to candidate compounds and monitored for the loss of polarised distribution of that tropomyosin isoform. The labelled tropomyosin isoform may be generated, for example, by expression of a fusion construct comprising tropomyosin linked to a fluorescent compound (such as the green fluorescent protein (GFP)) within the cell.

Those skilled in the art would understand that other detectable labels may be used in this screening assay.

Alternatively, a sample of cells may be exposed to a candidate compound, and the distribution of the tropomyosin isoform of interest determined by antibody staining.

Screening protocol based on expression of tropomyosin

An example of a screening method in which the ability of a candidate compound to inhibit tropomyosin expression may involve the following steps:

(i) contacting a candidate compound with cells capable of expressing tropomyosin,

(ii) measuring the amount of expression of tropomyosin in the cells brought into contact with the candidate compound and comparing this amount of expression with the amount of expression (control amount of expression) of tropomyosin in the corresponding control cells not brought into contact with an investigational substance, and

(iii) selecting a candidate compound showing a reduced amount of expression of tropomyosin as compared with the amount of control expression on the basis of the result of the above step (ii). The cells used in this screening method may be any cells that can express tropomyosin, irrespective of the difference between natural and recombinant genes. Moreover, the derivation of the tropomyosin 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. Examples of suitable human cells are hematopoietic cells including mast cells. Moreover, transformed cells that contain expression vectors comprising nucleic acid sequences that encode tropomyosin may also be used.

The conditions for allowing the candidate compound to come into contact with the cells that can express tropomyosin 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 tropomyosin genes can be expressed.

The term "reduced" refers not only the comparison with the control amount of expression, but also encompasses cases where no tropomyosin is expressed at all. Specifically, this includes circumstances wherein the amount of expression of tropomyosin is substantially zero.

The amount of expression of tropomyosin can be assessed either by measuring the amount of expression of a tropomyosin gene (mRNA) or by measuring the amount of a tropomyosin protein produced. In addition, the method to measure the amount of tropomyosin need not be a method to directly measure the amount of expression of gene (mRNA) or the amount of protein produced, but may be any method that reflects these.

Specifically, to measure the amount of expression of tropomyosin (detection and assay), the amount of expression of tropomyosin niRNA 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 tropomyosin mRNA. Moreover, the amount of tropomyosin protein may be measured by implementing such well-known methods as the Western blot method utilizing an anti-tropomyosin antibody.

The measurement of the amount of expression of tropomyosin (detection and assay) may be implemented by measuring the activity of proteins derived from marker genes, using a cell line into which have been introduced fused genes comprising the marker genes such as reporter genes (e.g., luciferase genes, chloramphenicol-acetyltransferase genes, β-glucuronidase genes, β-galactosidase genes and aequorin genes) linked to the tropomyosin gene. Alternatively, the expression of tropomyosin can be measured in a genetically engineered cell wherein a reporter sequence is introduced into the tropomyosin gene by homologous recombination so that the tropomyosin product expressed from that gene is labelled with the reporter.

Screening protocol based on binding of tropomyosin to one or more of its binding partners

In one embodiment, tropomyosin agonists or antagonists are identified by screening for candidate compounds which interfere with the binding of tropomyosin to a tropomyosin binding partner. An example of a suitable tropomyosin binding partner is actin.

Standard solid-phase ELISA assay formats are particularly useful for identifying antagonists of the protein-protein interaction. In accordance with this embodiment, one of the binding partners, e.g an actin filament, is immobilized on a solid matrix, such as, for example an array of polymeric pins or a glass support. Conveniently, the immobilized binding partner is a fusion polypeptide comprising Glutathione-S- transferase (GST; e.g. a CAP-actin fusion), wherein the GST moiety facilitates immobilization of the protein to the solid phase support. The second binding partner (e.g. tropomyosin) 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. Conveniently, 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. Alternatively, 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 tropomyosin to one of its binding partners. The basic mechanism of this system is similar to the yeast two hybrid system. In the two-hybrid system, the binding partners are expressed as two distinct fusion proteins in a mammalian host cell. In adapting the standard two-hybrid screen to the present purpose, a first fusion protein consists of a DNA binding domain which is fused to one of the binding partners, and 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 bioactive agents 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. On the other hand, 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.

Alternatively, 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 supra 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. 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. For example, a portion/fragment of tropomyosin that binds to actin is expressed as a DNA binding domain fusion, such as with the DNA binding domain of GAL4; and the portion of actin that binds tropomyosin is expressed as an appropriate transcription activation domain fusion polypeptide (e.g. with the GAL4 transcription activation domain). The fusion polypeptides are expressed in yeast in operable connection with the URA3 counter-selectable reporter gene, wherein expression of URA3 requires a physical relation between the GAL4 DNA binding domain and transcriptional activation domain. This physical relation is achieved, for example, by placing reporter gene expression under the control of a promoter comprising nucleotide sequences to which GAL4 binds. Cells in which the reporter gene is expressed do not grow in the presence of uracil and 5-fluororotic acid (5-FOA), because the 5-FOA is converted to a toxic compound. Candidate peptide inhibitor(s) are expressed in libraries of such cells, wherein cells that grow in the presence of uracil and 5-FOA are retained for further analysis, such as, for example, analysis of the nucleic acid encoding the candidate peptide inhibitor(s). Small molecules that antagonize the interaction are determined by incubating the cells in the presence of the small molecules and selecting cells that grow or survive of cells in the presence of uracil and 5-FOA.

Alternatively, a protein recruitment system, such as that described in U.S. Patent No. 5, 776, 689 to Karin et ah, may be used. In a standard protein recruitment system, 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. 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.

More specifically, the components of a protein recruitment system include a first expressible nucleic acid encoding a first fusion protein comprising the effector protein and one of the binding partners (e.g. actin or a portion thereof), and a second expressible nucleic acid molecule encoding a second fusion protein comprising a cell compartment localization domain and the other binding partner (e.g. tropomyosin 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), is also required, so that, in the absence of the protein-protein interaction, 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. Such a protein recruitment system can be practised in essentially any type of cell, including, for example, mammalian, avian, insect and bacterial cells, and using various effector protein/reporter molecule systems.

For example, a yeast cell based assay is performed, in which the interaction between tropomysoin 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. 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. To detect antagonists of the protein- protein interaction of the present invention, duplicate incubations are carried out in the presence 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 tropomysoin 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. For example, 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. In contrast, 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).

In yet another embodiment, small molecules are tested for their ability to interfere with binding of tropomyosin 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.

In a preferred embodiment of the invention the tropomyosin binding partner is selected from the group consisting of calponin (Childs et al. BBA 1121: 41-46, 1992),

Cancinoembryonic antigen cell adhesion molecule 1 (CEACAMl) (Schumann et ah, J.

Biol. Chem. 276 (50):47421-33, 2001), endostatin (MacDonald et al. J. Biol. Chem.

276, 25190-25196, 2001), Enigma (Guy et al. FEBS letters 10: 1973-1984, 1999),

Gelsolin (preferably sub-domain 2) Koepf and Burtnick FEBS 309(1): 56-58,, 1992), Sl 00 A2 (Gimona et al. J. Cell Sci. 110 : 611 -621 , 1997) and actin. In a further preferred embodiment, the tropomyosin binding partner is actin.

Screening method based on myosin ATPase activity

In an adaptation of the screening protocol based on binding of tropomyosin to one or more of its binding partners, the method involves the addition of myosin to the reaction mix and detection of myosin ATPase activity.

For example, a tropomyosin isoform may be incubated with actin filaments and specific myosins. Myosin ATPase activity is then measured in the presence of the candidate compounds. Under normal conditions, tropomyosin inhibits myosin ATPase activity.

Accordingly, compounds that interact with tropmyosin and prevent this inhibitory activity will results in increased myosin ATPase activity. Such compounds may be selected for further screening and/or characterisation. Suitable positive control reactions may be performed without tropomyosin or with an inappropriate tropomyosin isoform to eliminate anti-myosin effects.

Methods for determining myosin ATPase activity that can be adapted for use in the present invention will be known to those skilled in the art. Examples of such assays are described in Zhao et al., Biochem. Biophys. Res Commun. 267(l):77-79, 2000; Westra et al., Archives of Physiology and Biochemistry 109:316-322, 2001; and Drott et al., Biochem J. 264:191-8, 1989. Therapeutic methods

The tropomyosin agonists or antagonists identified by the methods of the present invention can be used therapeutically for antiproliferative diseases. The term "therapeutically" or as used herein in conjunction with the tropomyosin agonists or antagonists of the invention denotes both prophylactic as well as therapeutic administration. Thus, tropomyosin agonists/antagonists can be administered to high- risk patients in order to lessen the likelihood and/or severity of a disease or administered to patients already evidencing active disease.

Modes of Administration

In the case where 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. Generally speaking, the following 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. The oral preparations., for instance, 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 creams, 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 (pharmaceutical preparation) can be prepared by formulating the compound having tropomyosin 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. For example, 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 to about 100 mg, and this amount can be administered once a day or in a few divided doses daily.

When the substance having tropomyosin inhibitory activity is in the form of a polynucleotide such as an antisense compound, the composition may be provided in the form of a drug for gene therapy or a prophylactic drug. Recent years have witnessed a number of reports on the use of various genes, and gene therapy is by now an established technique.

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, viz. 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. Regarding the case in which a viral vector is used as said vector and the case in which a non-viral vector is used, respectively, 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.].

When using a non- viral vector, any expression vector capable of expressing the anti- tropomyosin nucleic acid may be used. Suitable examples include pCAGGS [Gene 108, 193-200 (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. By so doing, the object polynucleotide can be introduced into the patient's cells or tissue.

More particularly, 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, the 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, 19991]. For use in the prevention or treatment of an inflammatory disease to which the present invention is addressed, the drug is preferably introduced into the body by the in vivo technique.

When the in vivo method is used, the drug can be administered by a route suited to the object disease, target organ or the like. For example, 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. In the case of an injectable form, for instance, 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. In the case of liposomes such as HVJ-liposome, the drug can be provided in various liposome- entrapped preparations in such forms as suspensions, frozen preparations and centrifugally concentrated frozen preparations. Furthermore, in order that the gene may be easily localized in the neighborhood of the affected site, a sustained-release preparation (eg. a minipellet) may be prepared and implanted near the affected site or 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. about. about 100 mg. preferably about 0.001. about, about 10 mg. This amount is preferably administered several days or a few months apart.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application.

The present invention will now be illustrated by the following Examples, which are not intended to be limiting in any way.

Experimental Details

Materials and Methods

Antibodies

Tm proteins are encoded by 4 distinct genes. The antibodies used in this study were capable of detecting specific isoforms that are produced from three Tm genes. The exon/intron structure of these genes is shown in Figure 1. The mouse monoclonal LCl antibody specifically identifies TmhTM5NMl (Warren et al., 1995). The mouse monoclonal 311 antibody (Sigma Aldrich St. Louis, MO, U.S.A.) recognises the Ia exon in the amino terminus of both the α-Tm and β-Tm gene thus detecting Tms 1, 2, 3, and 6. The sheep monoclonal αf9d antibody recognises the 9d exon in the carboxyl terminus of the same genes and detects Tm 1, 2, 3, 5a, 5b and 6 (Schevzov et al., 1997). The CG3 antibody (a gift from J.C. Lin Univ. of Iowa, Iowa, U.S.A.) recognises the Ib exon of the γ-gene and detects Tm5NMl-l l (Novy et al., 1993, Cell Motility & the Cytoskeleton 25, 267-281; Dufour et al, 1998, Journal of Biological Chemistry 273, 18547-18555). The non-muscle actins were detected using either the sheep polyclonal γ-actin (Schevzov et al., in press) or mouse monoclonal β-actin (clone AC-74, Sigma Aldrich St. Louis, MO, U.S.A.) antibodies which detect distinct epitopes in the Class 1 non-muscle N-terminus.

Generation and Screening of Transgenic Animals

Human TΠI5_NMI and rat Tm3 cDNA under the control of the β-actin promoter was removed from vector sequences by digestion with KpriL and EcoRI (Roche Diagnostics). Fertilized eggs were collected from superovulated FVB/NJ females on the day of mating, injected with the DNA, and transferred to pseudopregnant ARC/5 females on the same day according to standard protocols (Hogan et al (1994). Manipulating the Embryo., Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press). Transgenic mice were screened by Southern blot analysis of DNA extracted from mouse tails, digested with Sad (Roche), and probed with a 1-kb Sad DNA fragment derived from the 5' flanking region of the human β-actin promoter. Wild type mice were nontransgenic FVB/NJ littermates.

Tissue extraction

Whole tissues were weighed and suspended in an appropriate volume of 5OmM Tris, pH 7.5 (1:0.02 tissue:buffer). Tissues were homogenized in a 1.5mL centrifuge tube using a plastic pestle and were then boiled at 95°C for 10 mins. This was repeated a further three times and the tissue was the centrifuged at 13,000rpm for lOmins to remove debris. Protein concentration was determined using a BCA protein assay kit (Pierce, Rockford, IL) as per manufacturer's instructions. Preparation of Detergent Soluble and Insoluble Fractions

Triton-soluble and -insoluble fractions were performed as previously described (Minamide et al (1997) J Biol. Chem. 272, 8303-8309). In brief, cultured cells were washed free of medium with four washes of 4°C PBS and lysed in 10 mM Tris, pH 7.5, 2 mM MgCl₂, 0.5 mM DTT, 2 mM EGTA, 1% Triton X-IOO, and 7.5% glycerol. The cells were scraped off the dish and transferred to a centrifuge tube. Soluble and insoluble fractions were prepared by centrifugation of the lysates at 17,000 x g_msκ for 20 min. Proteins were detected in each fraction by immunoblot analysis.

Immunoblot Analysis

Cells were harvested at 75% confluency for protein analysis and proteins were extracted using the method described in Wessel and Flugge (1984) Anal. Biochem. 138, 141-143. Protein concentrations were determined using a BCA protein assay kit (Pierce, Rockford, IL). Proteins were loaded onto 15% low bis acrylamide gels and transferred to Immobilon-P PVDF membrane (Millipore Corporation, Bedford, MA). PVDF blots were blocked with 5% low fat skim milk overnight. Primary antibodies were diluted in TTBS (100 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20). Blots were incubated with primary antibody for an hour at room temperature, followed by two 10-min washes with TTBS. The blots were incubated with HRP- conjugated secondary antibodies (Horse radish peroxidase (HRP) anti-mouse and anti-rabbit IgG were from Amersham Life Sciences (Buckinghamshire, U.K.) diluted in TTBS for an hour. After two 5-min washes with TTBS, the blots were incubated in equilibration buffer for 2 min (100 mM Tris, pH 9.5, 50 mM MgCl₂, 100 mM NaCl). The bands were detected using CSPD (Roche Diagnostics) and exposure to Fuji x-ray film. Bands were quantified using ImageQuant V3.3 densitometry software (Molecular Dynamics).

Cell fixation

NIH 3T3 cells were fixed for 20 min at 4°C in freshly prepared 4% paraformaldehyde. Three 5 minute washes in PBS were then performed before the cells were incubated with DAPI (Molecular Probes, Eugene, OR) to stain the nucleus. Fibroblasts were then mounted using VectaSheild (Vector Laboratories Inc. Burlingame, CA, USA) then analysed by confocal microscopy as described below. Fluorescence microscopy

Fluorescence was examined with a TCS SP2 confocal laser scanning upright microscope (Leica Microsystems, Wetzler, Germany) using a 63x oil emersion objective. The distribution of fluorophores was measured by scanning at 475nm for CFP and 527nm for YFP using 8 line and three frame averages to eliminate noise. Images were scanned sequentially in the xyz plane and images were constructed by overlaying sections taken at lμm steps from the apical to the basal region of the cells.

Construction of Plasmids

For stable transfections human TΠI5_NM_I and rat Tm3 cDNAs containing only the coding region were cloned into the pHβApr(sig-) vector under control of the human β-actin promoter (Temm-Grove et al. (1996) Cell Motil. Cytoskel. 33, 223-240; Qin and Gunning (19917) J Biochem. Biophys. Methods 36, 63-72; Bryce et al., 2003). For transient transfections human Tm5NM-l containing the 3' UTR untranslated region and human Tm3 cDNA was generated by PCR and subcloned in frame into the pE YFP-Cl and pECFP-Cl expression vectors (Clontech) to create the pEYFP- Tm5NM-l and pECFP-Tm3 fusion proteins (see Figure 5) as previously described (Percival et al., 2000). The sequence of the constructs was verified on an ABI 373 DNA automated sequencer using standard dye terminator technology (DNA Sequencing Facility, Westmead Millennium Institute).

Cell Culture and transfections

NlH 3T3 fibroblasts

NIH 3T3 fibroblasts were maintained at 50% confluence in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 2 mM glutamine (Invitrogen) at 37°C humidified with 5% CO₂. For microscopy, fibroblasts were grown on poly-L-lysine (Sigma, St Louis, MO)-coated coverslips and glass chamber slides (Nalge Nunc International, Roskilde, Denmark). Plasmids were transfected into NIH 3T3 fibroblasts with FuGENE 6 transfection reagent (Roche Diagnostics, Castle Hill, Australia). Cells were incubated with FuGENE 6/DNA complexes for 24 h to allow for optimal expression. B35 rat neuronal cells

B35 rat neuronal cells (Schubert et al (1974). Nature 249, 224-227) and all stably transfected clones were also maintained in DMEM supplemented with 10% FBS and 2 niM L-Glutamine at at 37°C humidified with 5% CO₂. Cells were transfected stably using Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions. Transfected clones were selected using 1% geneticin (Invitrogen) and clones were maintained in 0.5% geneticin (Invitrogen) (Bryce et al., 2003). For microscopy, cells were grown on poly-D-lysine (Sigma, St Louis, MO)-coated glass chamber slides (Nalge Nunc International, Roskilde, Denmark).

Drug Treatments

NIH 3T3 cells

Cytochalasin D (Sigma, St Louis, MO) was stored as a 2 mM stock solution in dimethyl sulfoxide (DMSO). NIH 3T3 cells plated onto glass chamber slides were grown to ~50% confluency and then incubated with 0-lOμg/mL Cytochalasin D for lhr and then fixed with 4% paraformaldehyde as described below.

B35 rat neuronal cells B35 cells were plated onto 30mm dishes once at -75% confluency they were incubated with 0-lOug/mL Cytochalasin D for lhr. Cells were then placed on ice and the treatment terminated by washing the cells with ice cold PBS. Triton soluble and triton insoluble fractions were prepared as described above. In both cases, to confirm the vehicle was having no adverse effects on the cells, control cells (Oμg/mL) were incubated with DMSO alone.

Example 1: The supply of cytoskeletal Tins are limiting for their stable accumulation.

To determine whether or not Tm levels are limiting within mammalian cells we analysed the expression levels of Tm5NMl and Tm3 in tissues from transgenic mice that carry a transgene producing additional mRNA encoding each of these isoforms. Protein levels were compared with control wild type mice of the same genetic background. Results show that the levels of accumulation of the cytoskeletal proteins Tm5NMl and Tm3 are elevated in tissues from the transgenic mice and this increase can more than double the size of the normal protein pool for that isoform (see example of mouse brain in Figure 2A). We conclude that the Tm5NMl and Tm3 protein pools are not saturated and the cells can stably accumulate additional protein if supply is increased.

This was separately tested by preventing the gamma-Tm gene from generating Tm5NMl and Tm5NM2 protein by knocking out the gamma gene 9d exon as shown in Figure 5. Analysis of mouse tissues carrying only one copy of the gamma-Tm gene with the 9d exon revealed that the levels of Tm5NMl and 2 were decreased by half (Figure 2B). We conclude that the supply of cytoskeletal Tm5NMl and 2 determines the level of stable accumulation of the proteins and that reduced supply in the case of a hemizygous knock-out cannot be compensated by altered translation or protein turnover to maintain the pool size at the level of the wild type.

Example 2: Increased Tm5NMl and TM3 levels alter their biochemical partitioning and influence distribution of the actin pool

Tm3 and hTm5NMi plasmids were stably transfected separately into B35 neuroepithelial cells. The resultant Tm protein expression and distribution could be monitored by the antibodies LCl₃ which specifically identifies hTm5_NMi, and 311, which identifies Tm3 (Figure 3). Expression and distribution of the endogenous non-muscle actins could be detected using the γ- and β-actin specific antibodies. Detergent extraction and centrifugation was performed to separate the triton-soluble fraction (presumably containing short actin filaments and associated proteins) and the triton-insoluble fraction (which would contain the larger more stable actin filaments and associated proteins). The ratio of Tm or actin found in the soluble versus insoluble fraction was determined . In the Tm3 cells there is an increase in the solubility of γ-actin and in the TmSNiVi₁ cells there is a decrease in the solubility of total and β-actin. This is in keeping with immunofluoresence data from these cells showing that Tm3 is found in what appears to be shorter, therefore more soluble filaments, involved in filopodia formation and the cells overexpressing Tm5NM-l have significant increase in stress fibre formation, which by their nature are more triton insoluble (Bryce et al., 2003). These results indicate that Tms present within the cell can influence the distribution of the actin pool and the types of filaments formed as both γ- and β-actin show a similar distribution to the Tm3 or TΠI5NMI isoform depending on the cell in which they are expressed. Example 3: Tm composition confers sensitivity of actin filaments to different actin altering drugs.

B35 rat neuroepithelial cells stably transfected with either Tm5NMl or Tm3 or control cells were treated with 0, 0.1, 1.0 and 10 μg/mL of Cytochalasin D for lhr and the organisation of the actin filaments and cell morphology in these cells was evaluated by examination of cells co-stained with Rhodamine-Phalloidin and an antibody which recognises the ends of actin filaments. Control and Tm3 cells were more sensitive to Cytochalasin D at all doses than that observed for the Tm5NMl transfectants as judged by retention of Phalloidin staining and maintenance of cell surface area. The same experimental design was applied to cells treated with 0, 0.1, 1.0 and 10 μM of Latrunculin A for lhr. The Tm5NMl cells showed the greatest resistance to the drug and retained both Phalloidin staining and surface area at the highest dose whereas actin filament organisation and surface area was greatly altered at 1.0 μM and only the Tm3 cells were impacted by 0.1 μM. We conclude that Tm isoforms can determine the response of the actin cytoskeleton to different actin altering drugs.

Example 4: TIΠ5NM_I and Tm3 containing filaments have different sensitivity to Cytochalasin D

NIH3T3 fibroblasts were cotransfected with YFP-hTm5NMl-3'UTR and CFP-hTm3 (constructs shown in Figure 4). 24 hours post transfection cells were treated with Cytochalasin D (0 - lOμg/ml) for lhr and the subcellular localisation of the Tms analysed by confocal microscopy. In control cells, treated with DMSO alone, both Tm5NM-l and Tm3 were found predominantly in stress fibres with some perinuclear localisation. When looking specifically at the stress fibres it was hard to distinguish the two Tm populations. However, there were subtle overall differences, with the Tm5NM-l appearing to be found more concentrated on the leading edges of the cell. Treatment with the drug Cytochalasin D caused distinct differences in the redistribution of the two populations of Tms. This was most apparent at the higher concentrations of Cytochalasin treatment (1 and lOμg/ml). Although there was some colocalisation of Tm5NM-l and Tm3 there were also distinct pools with the Tm3 being found more at the protruding edges of the cells and the Tm5NM-l more perinuclear. This suggests that the Tm5NM-l and Tm3 containing filaments have different sensitivity to actin destabilising drugs such as Cytochalasin D. Example 5: Generation of 9d knockout mouse

A 9d exon knock-out mouse was made using a knock-out construct in which the entire coding region, 3' splice site and most of the 3' untranslated region of the 9d exon of the gamma -Tm gene was removed. To achieve this, a neo cassette consisting of the neomycin resistance gene (neo) flanked by loxP sites was inserted between the BamHl site at the 3' end of the 9d exon and the EcoRl site at the 5' end within the untranslated region. 1.2kb and 2.6kb homologous regions from either side of the deleted region were used to make the construct (see Figure 5).

The construct was inserted into mouse ES cells and homologous recombinants detected by neomycin resistance and confirmed by Southern blotting. Correct clones were grown and injected into mouse blastocysts to create chimeras. Further breeding produced germ line transmission of the knocked-out allele. Mouse embryos which lacked both knock-out alleles were used to generate fibroblasts which were characterised for growth properties. The -/- fibroblast cultures were found to have increased numbers of multinucleated cells indicating a possible problem associated with cytokinesis. Subsequent analysis revealed that 20% of -/- cells were in the G2/M phase of the cell cycle compared with 11% for controls. This suggests that progression of G2/M cells to Go/Gl through cytokinesis has been compromised in the -/- cells and indicates that Tm5NMl/2 is required for normal passage through cytokinesis.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are readily apparent to those skilled in molecular biology or related fields are intended to be within the scope of the invention.

Claims

Claims:

1. A method of screening for an antiproliferative agent, the method comprising determining the activity or cellular location of one or more isoforms of cytoskeletal tropomyosin in the presence of a candidate compound, wherein alteration of the activity or cellular location of the one or more isoforms of cytoskeletal tropomyosin in the presence of the compound when compared to the absence of the compound indicates that the compound is a potential antiproliferative agent.

2. A method according to claim 1 wherein the isoform of cytoskeletal tropomyosin is a low molecular weight isoform.

3. A method according to claim 2 wherein the low molecular weight isoform is selected from the group consisting of Tm5a, Tm5b, Tm5NM-l, Tm5NM-2, Tm5NM-4, Tm5NM-5, Tm5NM-6, Tm5NM-7 and Tm4.

4. A method according to any one of claims 1 to 3, wherein alteration of the cellular location of an isoform of cytoskeletal tropomyosin is a result of loss of polarised distribution.

5. A method according to any one of claims 1 to 4 which comprises determining the activity or cellular location of at least first and second isoforms of cytoskeletal tropomyosin in the presence of a candidate compound, wherein alteration of the activity or cellular location of the first isoform but not the second isoform in the presence of the compound when compared to the absence of the compound indicates that the compound is a potential antiproliferative agent.

6. A method according to claim 5 wherein the first isoform is a low molecular weight isoform and the second isoform is a high molecular weight isoform.

7. A method according to claim 6 wherein the low molecular weight isoform is selected from the group consisting of Tm5a, Tm5b, Tm5NM-l, Tm5NM-2, Tm5NM-4, Tm5NM-5, Tm5NM-6, Tm5NM-7 and Tm4.

8. A method according to claim 6 or claim 7 wherein the high molecular weight isoform is selected from the group consisting of TmI, Tm2, Tm3 and Tm6.

9. A method of screening for an antiproliferative agent, the method comprising determining the expression levels of one or more isoforms of cytoskeletal tropomyosin in the presence of a candidate compound, wherein altered expression of the one or more isoforms in the presence of the compound when compared to the absence of the compound indicates that the compound is a potential antiproliferative agent.

10. A method according to claim 9 wherein the isoform of cytoskeletal tropomyosin is a low molecular weight isoform.

11. A method according to claim 10 wherein the low molecular weight isoform is selected from the group consisting of Tm5a, Tm5b, Tm5NM-l, Tm5NM-2, Tm5NM-4, Tm5NM-5, Tm5NM-6, Tm5NM-7 and Tm4.

12. A method according to any one of claims 9 to 11 wherein reduced tropomyosin expression of the one or more cytoskeletal tropomyosin isoforms in the presence of the compound indicates that the compound is a potential antiproliferative agent.

13. A method according to claim 11 or claim 12 which comprises determining the expression levels of at least first and second isoforms of cytoskeletal tropomyosin in the presence of a candidate compound, wherein alteration of the expression levels of the first isoform but not the second isoform in the presence of the compound when compared to the absence of the compound indicates that the compound is a potential antiproliferative agent.

14. A method according to claim 13 wherein the first isoform is a low molecular weight isoform and the second isoform is a high molecular weight isoform.

15. A method according to claim 14 wherein the low molecular weight isoform is selected from the group consisting of Tm5a, Tm5b, Tm5NM-l, Tm5NM-2, Tm5NM-4,

Tm5NM-5, Tm5NM-6, Tm5NM-7 and Tm4.

16. A method according to claim 14 or claim 15 wherein the high molecular weight isoform is selected from the group consisting of TmI, Tm2, Tm3 and Tm6.

17. A method of screening for an antiproliferative agent, the method comprising determining the nature of at least one expression product of one or more genes encoding cytoskeletal tropomyosin in the presence of a 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 antiproliferative agent.

18. A method according to claim 17 wherein the isoform of cytoskeletal tropomyosin is a low molecular weight isoform.

19. A method according to claim 18 wherein the low molecular weight isoform is selected from the group consisting of Tm5a, Tm5b, Tm5NM-l , Tm5NM-2, Tm5NM-4, Tm5NM-5, Tm5NM-6, Tm5NM-7 and Tm4.

20. A method according to any one of claims 17 to 19 wherein the expression product is mRNA and the method involves detecting alternative spliced forms of an mRNA transcript when expression of the gene occurs in the presence of the candidate compound.

21. A method according to any one of claims 17 to 19 wherein the expression product is a 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.

22. A method according to any one of claims 17 to 21 which comprises determining the nature of at least a first and second expression product of one or more genes encoding cytoskeletal tropomyosin in the presence of a candidate compound, wherein alteration of the nature of the first expression product but not the second expression product in the presence of the compound when compared to the absence of the compound indicates that the compound is a potential antiproliferative agent.

23. A method according to claim 22 wherein the first expression product encodes or consists of a low molecular weight isoform of cytoskeletal tropomyosin and the second expression product encodes or consists of a high molecular weight isoform of cytoskeletal tropomyosin.

24. A method according to claim 23 wherein the low molecular weight isoform is selected from the group consisting of Tm5a, Tm5b, Tm5NM-l, Tm5NM-2, Tm5NM-4, Tm5NM-5, Tm5NM-6, Tm5NM-7 and Tm4.

25. A method according to claim 23 or claim 24 wherein the high molecular weight isoform is selected from the group consisting of TmI, Tm2, Tm3 and Tm6.

26. A method of screening for an antiproliferative agent, the method comprising measuring the binding of one or more isoforms of cytoskeletal tropomyosin to one of its binding partners in the presence of a candidate compound, wherein an altered level of binding of the one or more isoforms to its binding partner in the presence of the compound when compared to the absence of the compound indicates that the compound is a potential antiproliferative agent.

27. A method according to claim 26 wherein the isoform of cytoskeletal tropomyosin is a low molecular weight isoform.

28. A method according to claim 27 wherein the low molecular weight isoform is selected from the group consisting of Tm5a, Tm5b, Tm5NM-l, Tm5NM-2, Tm5NM-4, Tm5NM-5, Tm5NM-6, Tm5NM-7 and Tm4.

29. A method according to any one of claims 26 to 28 wherein a reduced level of binding of the isoform of tropomyosin to its binding partner in the presence of the compound indicates that the compound is a potential antiproliferative agent.

30. A method according to any one of claims 26 to 29 wherein the tropomyosin binding partner is selected from the group consisting of calponin, CEACAMl, endostatin, Enigma, Gelsolin, S100A2 and actin.

31. A method according to claim 30 wherein the tropomyosin binding partner is actin.

32. A method according to any one of claims 26 to 31 which comprises determining the level of binding of at least first and second isoforms of cytoskeletal tropomyosin to their binding partners in the presence of a candidate compound, wherein alteration of the level of binding of the first isoform but not the second isoform in the presence of the compound when compared to the absence of the compound indicates that the compound is a potential antiproliferative agent.

33. A method according to claim 32 wherein the first isoform is a low molecular weight isoform and the second isoform is a high molecular weight isoform.

34. A method according to claim 33 wherein the low molecular weight isoform is selected from the group consisting of Tm5a, Tm5b, Tm5NM-l, Tm5NM-2, Tm5NM-4, Tm5NM-5, Tm5NM-6, Tm5NM-7 and Tm4.

35. A method according to claim 33 or claim 34 wherein the high molecular weight isoform is selected from the group consisting of TmI, Tm2, Tm3 and Tm6.

36. A method according to any one of claims 1 to 35 wherein the candidate compound is selected from the group consisting of a peptide, an antibody directed against tropomyosin, a small organic molecule, a sugar, a polysaccharide, an antisense compound directed against tropomyosin-encoding mRNA, an anti-tropomyosin catalytic molecule and small interfering RNA (RNAi) molecule that targets tropomyosin expression.

37. A method according to any one of claims 1 to 35 wherein the candidate agent is an antisense compound, a catalytic molecule or an RNAi molecule directed against tropomyosin-encoding mRNA.

38. A method according to any one of claims 1 to 37 which further comprises formulating the compound for administration to a human or a non-human animal.

39. A method for method for reducing or inhibiting proliferation of a cell, the method comprising contact the cell with an agent that modulates expression, location or activity of one or more isoforms of cytoskeletal tropomyosin.

40. A method for the treatment or prevention of a proliferative disease, the method comprising administering to a subject an agent that modulates expression, location or activity of one or more isoforms of cytoskeletal tropomyosin.

41. A method according to claim 39 or claim 40 wherein the agent is an antisense compound, a catalytic molecule or an RNAi molecule targeted specifically against exon Ib or 9d of the TPM 1 gene (SEQ ID NO:7) or exon Ib or 9d of the TPM 3 gene (SEQ ID NO:8).

42. A method according to claim 39 or claim 40 wherein the agent is an antisense compound, a catalytic molecule or an RNAi molecule targeted to the sequence AGCTCGCTGGAGGCGGTG (SEQ ID NO: 13).

43. A method according to claim 39 or claim 40 wherein the agent is an antisense compound comprising the sequence CACCGCCUCCAGCGAGCT (SEQ ID NO: 14).