WO2009046483A1 - Therapeutic protocol for the treatment or prevention of thrombocytopenia - Google Patents

Abstract

The specification describes the use of the c-Myb/p300 transcriptional regulatory complex or a component thereof, other than c-Myb alone, in the manufacture of a medicament for use in the treatment or prevention of thrombocytopenia in a subject. An example of a component other than c-Myb is p300 or a functional homolog thereof. It is proposed herein that inhibiting the function of the c-Myb/p300 complex including reducing levels of a component therein or of the ability of the complex to form promotes megakaryocytopoiesis and haematopoiesis. Down regulation of c-Myb or p300 or the interaction between these molecules in a subject enables recovery of platelet levels in subjects with or at risk of developing thrombocytopenia as a result of administration of cytotoxic including apoptosis inducing treatments. Accordingly, in some embodiments, the herein described medicaments and agents are for use in conjunction with cytotoxic cancer treatment agents to enhance their efficacy by ameliorating thrombocytopenia. In another aspect, the invention provides for the use of an agent which inhibits the formation, expression or activity of the c-Myb/p300 transcriptional regulatory complex or a component thereof, other than c-Myb alone, in the manufacture of a medicament in the treatment, prevention or amelioration of thrombocytopenia in a subject. In another aspect, mouse models and cells derived therefrom are described useful in the screening and testing of suitable medicaments.

Description

Therapeutic protocol for the treatment or prevention of thrombocytopenia

FIELD

The present invention relates generally to the field of therapeutic targets. More particularly, the present invention provides therapeutic targets in the manufacture or selection of medicaments for the treatment of thrombocytopenia and related conditions and/or symptoms associated therewith. Therapeutic protocols for thrombocytopenia also form part of the present invention.

BACKGROUND

Bibliographic details of the publications referred to in this specification are also collected at the end of the description.

Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in any country.

The development of small therapeutic agents is a major goal in the pharmaceutical industry. Such agents are potentially relatively inexpensive to manufacture and are less likely to induce adverse immunological responses. One of the difficulties, however, in small therapeutic molecule development is target selection. Many potential targets lack suitability due to their pleiotropic nature and/or due to the level of redundancy in a particular pathway.

One medical condition having potential life threatening consequences is a low platelet count, a condition known as thrombocytopenia. Thrombocytopenia can arise congenitally or following autoimmune or haematological diseases such as Myelodysplastic Syndrome (MDS) and Idiopathic Thrombocytopenic Purpura (ITP) and is associated with chronic liver disease and viral infections, including HIV/ AIDS.

Thrombocytopenia is also a significant side-effect of cytotoxic cancer treatments where episodes of bleeding can put patients at risk and can interrupt or compromise therapy. Patients undergoing extensive surgery often experience severe episodes of acute thrombocytopenia which is associated with increased mortality (Kuter and Begley, Blood 100: 3457-3469, 2002).

Platelet transfusion is currently the only effective treatment of thrombocytopenia. However, platelet transfusion is only a temporary treatment and can give rise to immunohematological side-effects and risk of infection which can be associated with morbidity and mortality.

Pharmaceutical intervention would provide a significant medical benefit in the treatment of thrombocytopenia. However, the use of cytokines such as erythropoietin (EPO) and interleukin-11 (IL-I l) has achieved only modest success in relation to red blood cells and platelets, respectively (Kuter and Begley 2002, supra).

There is a need to identify targets which are involved in the regulation of platelet production in order to develop pharmaceutical agents to treat thrombocytopenia.

SUMMARY

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

The present invention identifies a target for the development of medicaments useful in the treatment of thrombocytopenia. The "treatment" of thrombocytopenia includes the promotion of megakaryocytopoiesis and haematopoiesis. The targets include the transcriptional regulatory complex, c-Myb/p300, or a component thereof, other than c-Myb alone, such as p300. Inhibition of the activity of the c-Myb/p300 transcriptional regulatory complex is proposed herein to suppress thrombocytopenia and to promote megakaryocytopoiesis and haematopoiesis.

Hence, one aspect of the present invention contemplates the use of the c-Myb/p300 transcriptional regulatory complex or a component thereof, other than c-Myb alone, in the manufacture of a medicament for use in the treatment or prevention of thrombocytopenia in a subject.

The term "manufacture" includes selection or design of a medicament.

An example of a component other than c-Myb is p300 or a functional homolog thereof. It is proposed herein that inhibiting the function of the c-Myb/p300 complex including reducing levels of a component therein or of the ability of the complex to form promotes megakaryocytopoiesis and haematopoiesis.

Accordingly, another aspect of the present invention provides a method for the treatment or prophylaxis of a subject with thrombocytopenia or who is at risk of developing same the method comprising administering to the subject an amount of an agent effective to inhibit formation, expression or activity of the transcriptional regulatory complex c-Myb/p300 for - A -

a time and under conditions sufficient for promotion of platelet production.

Conditions resulting in thrombocytopenia contemplated herein include a congenital condition, an autoimmune and haematological disease such as MDS and ITP, cytotoxic cancer treatment, surgery including bone marrow transplantation.

Therapeutic protocols for the treatment of conditions associated with thrombocytopenia are encompassed herein including the treatment of congenital conditions, immunological and haematological conditions and cancer.

The inhibition of the c-Myb/p300 complex includes inhibiting the activity, expression or formation of the complex itself as well as inhibiting a component therein as well as reducing the levels of a component of the complex.

The agents and medicaments of the present invention include small chemical molecules, cytokines, membrane penetrating immune-like molecules, cell penetrating peptides and nucleic acid molecules. Hence, the agents may affect activity of the complex or component therein or expression of a gene encoding a component.

Combination therapy including the use of a c-Myb/p300 inhibiting agent or an agent which antagonizes a component therein as more fully described in the description and the use of one or more cytokines or platelet transfusion or other therapeutic modalities also form part of the present invention. Other treatments include platelet transfusion, administration of a c-Myb antagonist and/or administration of thrombopoietin and/or IL-11. Other treatments include cytotoxic cancer treatments.

Pharmaceutically compositions useful in the treatment of thrombocytopenia are also contemplated herein.

The above summary is not and should not be seen in any way as an exhaustive recitation of all embodiments of the present invention. BRIEF DESCRIPTION OF THE FIGURES

Some figures contain color representations or entities. Color photographs are available from the Patentee upon request or from an appropriate Patent Office. A fee may be imposed if obtained from a Patent Office.

Figures IA through C are graphical representations showing that a mutation in p300 causes suppression of MpI^{" "} thrombocytopenia. (A) Map of chromosomal location PU6. Markers found to be homozygous 129/Sv are shown in white, heterozygous in gray and homozygous C57BL/6 in black. The number of animals with each haplotype is shown below. PU6 was localized between D15AahA10 and D15AahA3. (B) Sequence of PCR- amplified genomic DNA from representative Plt6/Plt6, PH6/+ and wild-type mice showing a T to A mutation in exon 10 of Pltό mice resulting in a Tyr to Asn substitution at amino acid 630. (C) Model of the p300 KIX domain indicating the Plt6 mutation site (red) and potentially disrupted contacts (blue), as well as residues previously mutated (Kasper LH, Boussouar F, Ney PA, et al., Nature. 419: 738-743, 2002) in mice (Y631, A635 and Y639, green). Modelled using FUGUE (Shi J, Blundell TL, Mizuguchi K. J MoI Biol. 310: 243- 257, 2001) by homology with pdb file, IKDX, the solution structure of mouse CREB binding protein KIX domain.

Figures 2A through C are graphical representations showing expanded megakaryocytopoiesis in MpT'^~PU6 mutants. Elevated platelet counts in Plt6 homozygous (6/6, PU6/PU6 or ^QQ^nwph6) and heterozygous (6/+, PK6/+ or p3OO^plt(5/+) mice were accompanied by (A) increased numbers of megakaryocytes (megs), shown as number per microscopic field (x600, BM; x200, spleen), (B) megakaryocyte colony-forming cells (meg-CFC), shown as number per 2.5xlO⁴ bone marrow (BM) or 10⁵ spleen cells and (C) colony forming units-spleen (cfu-s), shown as number per 1.5x10⁵ donor bone marrow cells at days 12 or 8 following transplantation. Means ± standard deviations are shown. * p<0.05 for comparison of data from MpI ^~'^~ PU6/PU6 or MpI ^''^' PH6/+ mice with that of MpI ^"Λ +/+ mice. n=3-7 mice per genotype. Figure 3 is a graphical representation showing response of c-Myb^PM/+ mice to 5-fluorouracil (5-FU). n=2-l l mice per point; *p<0.017, paired t-test c-Myb^PM/+ vs c-

_LPlt4/+ Myb^+/+; -O- c-Myb^+/+; -Q- c-Myb^F

Figure 4 is a graphical representation showing response of p30(f ^{t6 +} mice to 5-FU. n=2-5 mice per point; *p<0.017, paired t-test _v3QQ^plt6/+ vs p300^+/+; -O-p300^+/+; -Δ- p30(f^{lt6H '}.

Figure 5 is a graphical representation showing the response of c-Myb^PM/+ mice to bone marrow (BM) transplantation. n=2-6 mice per point; *p.O17, paired t-test c-Myb^pw/+ vs c- Myb^+/+; -O- c-Myb^+/+, -Q c-Myb^Plt4/+.

Figure 6 is a graphical representation showing the response of p30(f^lt6/+ mice to BM transplantation. n=3-7 mice per point; *p<0.017, paired t-test \>3§0^PM/* vs p300^+/+; -O- p300^+/+; -Δ- p300^plt6/+.

Figures 7A through C are representations of data from binding studies showing that a mutation in p300 KIX domain reduces p300 affinity for c-Myb. 2μg GST, GST-p300 KIX (Y630N) and GST-p300 KIX (wild-type) were used as baits for pulldown experiments with ³⁵S-labelled, in vitro transcribed/translated c-Myb. Pulldown reactions were resolved by SDS-PAGE followed by detection of bound ³⁵S-c-Myb by autoradiography. (A) Typical autoradiogram. (B) Coomassie Blue staining to determine bait levels. (C) The amount of bound ³⁵S-c-Myb was determined by densitometric analysis of the autoradiographs of three independent experiments and the ³⁵S-c-Myb bound by GST and GST-p300 KIX (Y630N) is shown relative to the amount bound by GST-p300 KIX (wild-type). Data represents the mean of three independent experiments with error bars corresponding to standard deviation.

Figure 8 are graphical representations showing expanded megakaryocytopoiesis in recipients of Mpl^+/+ _P30(f^lt6/Plt6 bone marrow. Recipients of Mpt^/+ p30(f^lt6/plt6 (6/6) and Mpl^+/+ p300^+/+ control (+/+) bone marrow exhibited (A) increased numbers of megakaryocytes (megs), shown as number per microscopic field (x600, BM; x200, spleen), and (B) megakaryocyte colony-forming cells (meg-CFC), shown as number per 2.5xlO⁴ bone marrow (BM) or 10⁵ spleen cells. Means ± standard deviations are shown. * p<0.05 for comparison of data from recipients of Mpl^+/+ p30(f^{lt6 P t6} marrow with that of MpI^{+ +} p300^+/+ marrow recipients. n=4 mice per genotype.

Figure 9 are graphical representations showing a B lymphocyte deficit in recipients of Mpl^+/+ p30(f^lt6/plt6 bone marrow. (A) Flow cytometry plots of bone marrow, lymph node and spleen cells from recipients of Mpl^+/+ p300^plt6/ph6 {PU6/PU6) and Mpl^+/+ p300^+/+ control (+/+) bone marrow stained with antibodies to B220 and IgM showing reduced proportions of B220⁺ cells, including IgM⁺ B cells in each tissue. (B) Percentages of B220⁺ cells in bone marrow (BM), spleen (spl), mesenteric lymph node (LN) and blood of recipients of Mpl^+/+ p30(f"^6/p"⁶ (PU6/PU6) and Mpl^+/+ p300^+/+ control (+/+) bone marrow. Means ± standard deviations are shown. * p<0.05 for comparison of data from recipients of Mpf^/+ p30(f^lt6/PU6 marrow with that of Mpl^+/+ p300^+/+ marrow recipients. n=4 mice per genotype.

DETAILED DESCRIPTION

The present invention is predicated in part upon the discovery that the transcriptional regulatory complex comprising c-Myb/p300 has an indispensable role in repressing megakaryocyte development, the down regulation of which allows substantial TPO- independent platelet production. In accordance with the present invention, inhibition or down regulation of the level of p300 or of a complex comprising p300 and c-Myb results in elevated levels of megakaryocytes, myeloid and megakaryocyte progenitor cells and platelets in mammalian cells. Mice bearing a mutation in the p300 KJX domain (Y630N) show reduced c-Myb binding and an enhanced ability to produce megakaryocytes and platelets.

The subject invention is not limited to particular screening procedures for agents, specific formulations of agents and various medical methodologies, as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

Each embodiment described in this specification is to be applied mutatis mutandis to every other embodiment unless expressly stated otherwise.

As used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a medicament" includes a single medicament, as well as two or more medicaments; reference to "an agent" includes a single agent as well as two or more agents; reference to "the invention" includes a single aspect or multiple aspects of an invention; and so on.

The term "subject" as used herein refers to an animal, in particular a mammal and more particularly a primate including a lower primate and even more particularly, a human who can benefit from the medical protocol of the present invention. A subject regardless of whether a human or non-human animal or embryo may be referred to as an individual, subject, animal, patient, host or recipient. The present invention has both human and veterinary applications. For convenience, an "animal" specifically includes livestock animals such as cattle, horses, sheep, pigs, camelids, goats and donkeys. With respect to horses, these include horses used in the racing industry as well as those used recreationally or in the livestock industry.

Examples of laboratory test animals include mice, rats, rabbits, guinea pigs and hamsters. Rabbits and rodent animals, such as rats and mice, provide a convenient test system or animal model as do primates and lower primates. Particularly useful test animals are referred to as PU4/+ mice and PU6/+ mice as well as their homozygous forms alone or together with MpT'^' mice. These are described in more detail below.

The terms "disorder", "abnormality" and "condition" may be used interchangeably to refer to an adverse health condition brought about by the functioning or activity of the transcriptional regulatory complex, c-Myb/p300. As described herein, this complex is associated with megakaryocyte development. It is proposed herein that the inhibition of the c-Myb/p300 transcriptional complex enables thrombopoietin (TPO)-independent platelet production. Hence, the c-Myb/p300 transcriptional complex is proposed herein to be a target for the development and screening of antagonists or other agents useful in promoting megakaryocytopoiesis and hemopoiesis and TPO-independent platelet production. The present invention extends to an individual component of the c-Myb/p300 complex as a target with the exception of c-Myb alone. Antagonists of c-Myb may, however, be used in connection with antagonists of other components of the c-Myb/p300 complex and/or antagonists of the c-Myb/p300 complex itself.

The terms "modulate", "inhibit" or "down regulate" include antagonising, decreasing, reducing and partially inhibiting formation, expression or activity of the transcriptional regulatory complex comprising c-Myb/p300 or a component thereof, other than c-Myb alone in relation to enhancing the ability of a subject to produce platelets.

Hence, one aspect of the present invention contemplates the use of the c-Myb/p300 transcriptional regulatory complex or a component thereof, other than c-Myb alone, in the manufacture of a medicament in the treatment of thrombocytopenia in a subject. In some embodiments, components of the c-Myb/p300 transcriptional regulatory complex include not only c-Myb and p300 but one or more of CREB, Tax, ReIA, JUN, SREBP-2, Stat2, E2F-1, p53, MDM2, SYT, HIFl, RXR, ER, RAR, GR, TR, PR, JMY, ElA, SV40 t antigen, NAP, pCAF, MyoD, FOS, p73, PP90 RSK, TBP, TF₁₁B, YY-I, CBP, SRC-I or a functional fragment thereof (see for example, Chan & La Thangue, J. Cell Science, 114: 2363-2373, 2001. Also included are Pinl isomerase (Pani et al., Biochim Biophys Acta. Jun; 1783(6): 1121-8, 2008), Mi-2alpha (Saether et al., J. Biol. Chem. May 11; 282(19): 13994-4005, 2007), histone H3 tails (Mo et al, Genes Dev. Oct 15; 19(20): 2447-57, 2005), AFTBl (Kaspar et al, J Biol Chem. May 14; 274(20): 14422-8, 1999), and c-Maf (Hedge et al, MoI Cell Biol. May; 18(5): 2729-37, 1998). Further components of the c-Myb/p300 complex can be identified using methods well known in the art, such as using yeast-2-hybrid screening and proteomics techniques, for example, two-dimensional gel electrophoresis and mass spectrometry.

Reference to "manufacture" includes selection and design of medicaments.

Reference to "thrombocytopenia" includes any condition leading to a deficiency of platelets or a condition associated with a low platelet count. The conditions may be congenital or arise following autoimmune or haematological disease such as Myelodysplastia Syndrome (MDS) or Idiopathic Thrombocytopenia (ITP). Thrombocytopenia may also arise as a side-effect of cytotoxic cancer treatment, especially where episodes of bleeding put patients at risk and also interrupt and compromise therapy. Patients undergoing extensive surgery can also experience thrombocytopenia which is associated with mortality and morbidity.

The inhibition of activity or the disruption of the c-Myb/p300 transcriptional complex is proposed herein to enhance megakaryocytopoiesis and haematopoiesis.

Hence, another aspect of the present invention provides for the use of the c-Myb/p300 transcriptional regulatory complex or a component thereof, other than c-Myb alone, in the manufacture of a medicament to induce or otherwise facilitate megakaryocytopoiesis or haematopoiesis in a subject. In some embodiments, the component of c-Myb/p300 is p300 or a functional homolog thereof.

Still another aspect of the present invention is directed to the use of the c-Myb/p300 transcriptional regulatory complex or a component thereof, other than c-Myb alone, in the manufacture of a medicament to induce or otherwise facilitate production of platelets in a subject, hi some embodiments, the component of c-Myb/p300 is p300 or a functional homolog thereof.

Yet still another aspect of the present invention provides for the use of the c-Myb/p300 transcriptional regulatory complex or a component thereof, other than c-Myb alone, in the manufacture of a medicament to induce or otherwise facilitate treatment of conditions resulting in a low platelet count in a subject, hi some embodiments, the component of c- Myb/p300 is p300 or a functional homolog thereof.

In yet another aspect the present invention provides for the use of an agent which inhibits the formation, expression or activity of the c-Myb/p300 transcriptional regulatory complex or a component thereof, other than c-Myb alone, in the manufacture of a medicament to induce or otherwise facilitate megakaryocytopoiesis or haematopoiesis in a subject, hi other embodiments, it is proposed to use the medicament to induce or otherwise facilitate production of platelets in a subject. In yet other embodiments, it is proposed to use the medicament to induce or otherwise facilitate treatment of conditions resulting in low platelet count in a subject, hi a preferred embodiment, the subject is a human.

Both the activity of the c-Myb/p300 complex and the formation of the complex may be the target for antagonism as may be the activity or level of an individual component. Hence, the transcriptional activity may be inhibited or the action of a particular component of the complex is inhibited or its level reduced or its ability to participate in the complex may be inhibited by the agents described herein. Such agents can be described herein as "c-Myb/p300 antagonists" or "cM/P antagonists" or "cM/PAs" and all such agents include antagonists of activity, levels, expression of genes encoding a component and the ability of a component to participate in complex formation. The present invention provides cM/PAs for use in the treatment of thrombocytopenia or conditions which result in or have the potential to result in thrombocytopenia. Such treatment or prophylaxis includes promotion of megakaryocytopoiesis and haematopoiesis.

In some embodiments, accordingly, the present invention provides for the use of an agent which inhibits the formation, expression or activity of the c-Myb/p300 transcriptional regulatory complex or a component thereof, other than c-Myb alone, in the manufacture of a medicament in the treatment of thrombocytopenia in a subject. In an illustrative embodiment, the agent is a small molecule, an antibody, a nucleic acid or a protein or peptide, such as a stapled peptide.

Agents which have the potential to act as c-Myb/p300 antagonists include small chemical molecules which can penetrate a cell membrane or via an ion channel or other pore and an antigen binding agent which has the capacity for intracellular transmission such as cartilage fish-derived antibodies (e.g. shark antibodies; see for example, Liu et al., BMC Biotechnol. 7: 78, 2007). An antigen binding agent, or functionally active fragment thereof, which has the capacity for intracellular transmission also includes antibodies such as camelids and llama antibodies, scFv antibodies, intrabodies or nanobodies, e.g. scFv intrabodies and V_11H intrabodies. Such antigen binding agents can be made as described by Harmsen & De Haard in Appl. Microbiol. Biotechnol. Nov; 77(1): 13-22, 2007; Tibary et al, Soc. Reprod. Fertil. Suppl. 64: 297-313, 2007; Muyldermans, J. Biotechnol. 74: 277- 302, 2001; and references cited therein. In one embodiment, scFv intrabodies which are able to interfere with a protein-protein interaction are used in the methods of the invention; see for example, Visintin et al, J. Biotechnol, 755:1-15, 2008 and Visintin et al, J. Immunol. Methods, 290(1-2): 135-53, 2008 for methods for their production. For use in the methods of the invention, such agents may comprise a cell-penetrating peptide sequence or nuclear-localizing peptide sequence such as those disclosed in Constantini et al., Cancer Biotherm. Radiopharm. 23(1): 3-24, 2008. Also useful for in vivo delivery are Vectocell or Diato peptide vectors such as those disclosed in De Coupade et al., Biochem J. 39(Hpt2): 407-418, 2005 and Meyer-Losic et al, J Med Chem. 49(23): 6908-6916, 2006. Thus, the invention provides the therapeutic use of fusion proteins of the agents (or functionally active fragments thereof), for example but without limitation, where the antibody or fragment thereof is fused via a covalent bond (e.g. a peptide bond), at optionally the N-terminus or the C-terminus, to a cell-penetrating peptide or nuclear- localizing peptide sequence.

Some suitable peptide agents that antagonize p300 are known in the art and are described for example in Frangioni et al. Nature Biotechnology, 18: 1080-1085, 2000 and Rutledge et al. J Am Chem Soc. 125: 14336-14347, 2003.

Natural products, combinatorial synthetic organic or inorganic compounds, peptide/polypeptide/protein, nucleic acid molecules and libraries or phage or other display technology comprising these are all available to screen or test for suitable agents. Natural products include those from coral, soil, plant, or the ocean or Antarctic environments. Libraries of small organic molecules can be generated and screened using high-throughput technologies known to those of skill in this art. See for example United States Patent No. 5,763,623 and United States Application No. 20060167237. Combinatorial synthesis provides a very useful approach wherein a great many related compounds are synthesized having different substitutions of a common or subset of parent structures. Such compounds are usually non-oligomeric and may be similar in terms of their basic structure and function, for example, varying in chain length, ring size or number or substitutions. Virtual libraries are also contemplated and these may be constructed and compounds tested in silico (see for example, US Publication No. 20060040322) or by in vitro or in vivo assays known in the art. Libraries of small molecules suitable for testing are already available in the art (see for example, Amezcua et al, Structure (London) 10: 1349-1361, 2002). Yeast SPLINT antibody libraries are available for testing for intrabodies which are able to disrupt protein-protein interactions (see Visintin et al., supra). Examples of suitable methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al, Proc. Natl. Acad. Sci. USA 90: 6909, 1993; Erb et al, Proc. Natl. Acad. Sci. USA 91: 11422, 1994; Zuckermann et al, J. Med. Chem. 37: 2678, 1994; Cho et al, Science 261: 1303, 1993; Carrell et al, Angew. Chem. Int. Ed. Engl. 33: 2059, 1994; Carell et al, Angew. Chem. Int. Ed. Engl. 33: 2061, 1994; and Gallop et al, J. Med. Chem. 37: 1233, 1994.

Thus, agents can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is suited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, Anticancer Drug Des. 12: 145, 1997; United States Patent No. 5,738,996; and United States Patent No. 5,807,683). Libraries of compounds may be presented, for example, in solution (e.g. Houghten, Bio/Techniques 13: 412-421, 1992), or on beads (Lam, Nature 354: 82-84, 1991), chips (Fodor, Nature 364: 555-556, 1993), bacteria (United States Patent No. 5,223,409), spores (United States Patent Nos. 5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al, Proc. Natl. Acad. Sci. USA 89: 1865- 1869, 1992) or phage (Scott and Smith, Science 249: 386-390, 1990; Devlin, Science 249: 404-406, 1990; Cwirla et al, Proc. Natl. Acad. Sci. USA 87: 6378-6382, 1990; and Felici, J. MoI. Biol. 222: 301-310, 1991).

In addition, genetic molecules and viruses comprising same are used to induce silencing of a gene encoding p300 or other component of the c-Myb/p300 complex. Nucleic acids including DNA (gDNA, cDNA), RNA (sense RNAs, antisense RNAs, mRNAs, tRNAs, rRNAs, small interfering RNAs (SiRNAs), double-stranded RNAs (dsRNA), short hairpin RNAs (shRNAs), piwi-interacting RNAs (PiRNA), micro RNAs (miRNAs), small nucleolar RNAs (SnoRNAs), small nuclear (SnRNAs) ribozymes, aptamers, DNAzymes or other ribonuclease-type complexes are conveniently employed. In particular, such nucleic acids are described in United States Patent Application No. 20050159378 incorporated herein. In particular, RNA interference of Myb is described therein. Corresponding molecules may be identified for p300 using similar procedures. Methods of producing chimeric constructs capable of producing dsRNA in eukaryotic cells are also described in the art.

Aptamers are also contemplated. RNA and DNA aptamers can substitute for monoclonal antibodies in various applications (Jayasena, Clin. Chem., 45(9): 1628-1650, 1999; Morris et al, Proc. Natl. Acad. Sci., USA, 95(6): 2902-2907, 1998). Aptamers are nucleic acid molecules having specific binding affinity to non-nucleic acid or nucleic acid molecules through interactions other than classic Watson-Crick base pairing. Aptamers are described, for example, in United States Patent Nos. 5,475,096; 5,270,163; 5,589,332; 5,589,332; and 5,741,679. An increasing number of DNA and RNA aptamers that recognize their non- nucleic acid targets have been developed by SELEX and have been characterized (Gold et al, Annu. Rev. Biochaem., 64: 763-797.1995; Bacher et al, Drug Discovery Today, 5(6): 265-273, 1998).

In some embodiments, agents that down modulate the formation, expression or activity of c-Myb/p300 may be derived from c-Myb or p300 or their encoding sequences or are variants or analogs thereof. Thus, for example, agents may be hydrocarbon-stapled peptides or minature proteins which are alpha-helical and cell-penetrating, and are able to disrupt protein-protein interactions (see for example, Wilder et al, ChemMedChem. 2(8):

1149-1151, 2007; & for a review, Henchey et al, Curr. Opin. Chem. Sept 12, 2008). In a particular embodiment, the agent is a hydrocarbon-stapled peptide based upon the sequence of the KIX domain of p300.

In some embodiments, the agents are derived from nucleic acid molecules such as the nucleotide sequences of c-Myb or p300 as described herein or corrected version thereof or variants thereof. Variants include nucleic acid molecules sufficiently similar to naturally occurring forms of these molecules or their complementary forms over all or part thereof such that selective hybridisation may be achieved under conditions of medium or high stringency, or which have about 60% to 90% or 90 to 98% sequence identity to the nucleotide sequences defining naturally occurring c-Myb or p300 sequences as described herein and over a comparison window comprising at least about 15 nucleotides. Preferably the hybridisation region is about 12 to about 18 nucleobases or greater in length. Preferably, the percent identity between a particular nucleotide sequence and the reference sequence is at least about 80%, or 85%, or more preferably about 90% similar or greater, such as about 95%, 96%, 97%, 98%, 99% or greater. Percent identities between 80% and 100% are encompassed. The length of the nucleotide sequence is dependent upon its proposed function. For example, short interfering RNAs are generally about 20 to 24 nucleotides in length, whereas molecules designed to provide dominant negative functions may require full length or substantially full length molecules. The term "homolog" or "homologs" refers broadly to functionally and structurally related molecules including those from other species. Homologs and orthologs are examples of variants.

In some embodiments the present invention contemplates the use of full length c-Myb or p300 polypeptides or biologically active portions or stapled peptides of one or more of these molecules as antagonists. Biologically active portions or stapled peptides comprise one or more binding domains. A biologically active portion or stapled peptide of a full length polypeptide can be a polypeptide which is, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 300, 350, 400, 450, 500, 550, 600 to about 640 and in the case of p300 which comprises about 2420 amino acids, 700, 800, 900, 1000, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400 to about 2410 or more amino acid residues in length. The c-Myb or p300 polypeptides contemplated include all biologically active or naturally occurring forms of c-Myb or p300 as well as biologically active portions and variants thereof.

"Variant" polypeptides include proteins derived from the native protein by deletion (so- called truncation) or addition of one or more amino acids to the N-terminal and/or C- terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present invention are biologically active, that is, they continue to possess at least one biological activity of the native protein. Antagonist variants are selected on the basis that they inhibit or antagonise the biological activity or formation of the cMyb/p300 or a component thereof. Such variants may result from, for example, genetic polymorphism or from human manipulation. Biologically active variants of a native p300 or c-Myb polypeptide will have at least 40%, 50%, 60%, 70%, generally at least 75%, 80%, 85%, preferably about 90% to 95% or more, and more preferably about 98% or more sequence similarity with the amino acid sequence for the native protein as determined by contemporary sequence alignment programs using default parameters. A biologically active variant of a p300 or c-Myb polypeptide may differ from that polypeptide generally by as much 100, 50 or 20 amino acid residues or suitably by as few as 1-15 amino acid residues, as few as 1-10, such as 6- 10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.

A p300 or c-Myb polypeptide/peptide may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a p300 or c-Myb polypeptides can be prepared by introducing mutations in the encoding DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (Proc. Natl. Acad. Sci. USA, 82: 488-492, 1985), Kunkel et al, (Methods in Enzymol., 154: 367-382, 1987), United States Patent No. 4,873,192, Watson et al. ("Molecular Biology of the Gene", Fourth Edition, Benjamin/Curnmings, Menlo Park, Calif., 1987) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al, (Natl. Biomed. Res. Found, 5: 345-358, 1978). Methods for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property are known in the art. Such methods are adaptable for rapid screening of the gene libraries generated by combinatorial mutagenesis of polypeptides. Recursive ensemble mutagenesis (REM), a technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify useful polypeptide variants (Arkin et al., Proc. Natl. Acad. Sci. USA, 89: 7811-7815, 1992; Delgrave et al, Protein Engineering, 6: 327-331, 1993). Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be desirable as discussed in more detail below. Variant p300 or c-Myb polypeptides may contain conservative amino acid substitutions at various locations along their sequence, as compared to reference amino acid sequences. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, which can be generally sub- classified as follows:

Acidic: The residue has a negative charge due to loss of H ion at physiological pH and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH. Amino acids having an acidic side chain include glutamic acid and aspartic acid.

Basic: The residue has a positive charge due to association with H ion at physiological pH or within one or two pH units thereof (e.g., histidine) and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH. Amino acids having a basic side chain include arginine, lysine and histidine.

Charged: The residues are charged at physiological pH and, therefore, include amino acids having acidic or basic side chains (i.e., glutamic acid, aspartic acid, arginine, lysine and histidine).

Hydrophobic: The residues are not charged at physiological pH and the residue is repelled by aqueous solution so as to seek the inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium. Amino acids having a hydrophobic side chain include tyrosine, valine, isoleucine, leucine, methionine, phenylalanine and tryptophan. As shown herein, substitution of tyrosine for asparagine in the KIX domain of p300 (Y630N) profoundly alters its ability to bind c-Myb. Neutral/polar: The residues are not charged at physiological pH, but the residue is not sufficiently repelled by aqueous solutions so that it would seek inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium. Amino acids having a neutral/polar side chain include asparagine, glutamine, cysteine, histidine, serine and threonine.

This description also characterises certain amino acids as "small" since their side chains are not sufficiently large, even if polar groups are lacking, to confer hydrophobicity. With the exception of proline, "small" amino acids are those with four carbons or less when at least one polar group is on the side chain and three carbons or less when not. Amino acids having a small side chain include glycine, serine, alanine and threonine. The gene-encoded secondary amino acid proline is a special case due to its known effects on the secondary conformation of peptide chains. The structure of proline differs from all the other naturally-occurring amino acids in that its side chain is bonded to the nitrogen of the a- amino group, as well as the α-carbon. Several amino acid similarity matrices (e.g., PAM120 matrix and PAM250 matrix as disclosed for example by Dayhoff et al., 1978 (supra); and by Gonnet et al, Science, 256(5062): 1443-1445, 1992), however, include proline in the same group as glycine, serine, alanine and threonine. Accordingly, for the purposes of the present invention, proline is classified as a "small" amino acid.

Amino acid residues can be further sub-classified as cyclic or noncyclic, and aromatic or nonaromatic, self-explanatory classifications with respect to the side-chain substituent groups of the residues, and as small or large. The residue is considered small if it contains a total of four carbon atoms or less, inclusive of the carboxyl carbon, provided an additional polar substituent is present; three or less if not. Small residues are, of course, always nonaromatic. Dependent on their structural properties, amino acid residues may fall in two or more classes. For the naturally-occurring protein amino acids, sub-classification according to this scheme is presented in the Table 5.

Conservative amino acid substitution also includes groupings based on side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. For example, it is reasonable to expect that replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting variant polypeptide. Whether an amino acid change results in a functional c-Myb or p300 polypeptide can readily be determined by assaying its activity. Activities that can readily be assessed are known to those of skill and include assays to determine binding or dimerization or oliomerization detected by, for example, nuclear magnetic resonance spectroscopy (NMR) where heteronuclear single quantum coherence (HSQC) spectra are observed, Biacore, kinetic, affinity and pull-down analyses. Conservative substitutions are shown in Table 6 below under the heading of exemplary substitutions. More preferred substitutions are shown under the heading of preferred substitutions. Amino acid substitutions falling within the scope of the invention, are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. After the substitutions are introduced, the variants are screened for biological activity.

Alternatively, similar amino acids for making conservative substitutions can be grouped into three categories based on the identity of the side chains. The first group includes glutamic acid, aspartic acid, arginine, lysine, histidine, which all have charged side chains; the second group includes glycine, serine, threonine, cysteine, tyrosine, glutamine, asparagine; and the third group includes leucine, isoleucine, valine, alanine, proline, phenylalanine, tryptophan, methionine, as described in Zubay, G., Biochaemistry, third edition, Wm.C. Brown Publishers (1993). Thus, a predicted non-essential amino acid residue in a p300 or c-Myb polypeptide is typically replaced with another amino acid residue from the same side chain family. Alternatively, mutations can be introduced randomly along all or part of the polynucleotide coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for an activity of the parent polypeptide to identify mutants which retain that activity. Following mutagenesis of the coding sequences, the encoded peptide can be expressed recombinantly or produced synthetically and the activity of the peptide can be determined.

Accordingly, the present invention also contemplates variants of the naturally-occurring c-Myb or p300 polypeptide sequences or their biologically-active fragments, wherein the variants are distinguished from the naturally-occurring sequence by the addition, deletion, or substitution of one or more amino acid residues. In general, variants will display at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 % identity to a reference c-Myb or p300 polypeptide sequence as described herein. Moreover, sequences differing from the native or parent sequences by the addition, deletion, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 or more amino acids but which retain certain properties of the reference p300 or c-Myb polypeptide are contemplated. The present variant p300 or c-Myb polypeptides also include polypeptides that are encoded by polynucleotides that hybridize under stringency conditions as defined herein, especially high stringency conditions, to p300 or c-Myb polynucleotide sequences, or the non-coding strand thereof.

In some embodiments, variant polypeptides differ from a c-Myb or p300 polypeptide sequence by at least one but by less than 50, 40, 30, 20, 15, 10, 8, 6, 5, 4, 3 or 2 amino acid residue(s). In another, variant polypeptides differ from the corresponding sequence in any referenced sequence by at least 1% but less than 20%, 15%, 10% or 5% of the residues. If this comparison requires alignment the sequences should be aligned for maximum similarity. ("Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) In one embodiment, the differences are differences or changes at a non-essential residue or a conservative substitution. A sequence alignment for c-Myb or p300 proteins from a range of mammalian species is used to demonstrate conserved residues.

A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of an embodiment polypeptide without abolishing or substantially altering one or more of its activities. Suitably, the alteration does not substantially alter one of these activities, for example, the activity is at least 20%, 40%, 60%, 70% or 80% of wild-type.

An "essential" amino acid residue is a residue that, when altered from the wild-type sequence of an polypeptide agent of the invention, results in abolition of an activity of the parent molecule such that less than 20% of the wild-type activity is present.

hi other embodiments, a variant polypeptide includes an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98% or more similarity to a corresponding sequence of a c-Myb or p300 polypeptide as described herein, and has at least one activity of that p300 or Myb polypeptide.

In some embodiments, analogs of antagonists of p300 polypeptides have enhanced stability and activity or reduced unfavourable pharmacological properties. They may also be designed in order to have an enhanced ability to cross biological membranes or to interact with only specific substrates. Thus, analogs may retain some functional attributes of the parent molecule but may posses a modified specificity or be able to perform new functions useful in the present context i.e., for administration to a subject. Analogs of peptide or polypeptide agents contemplated herein include but are not limited to modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogs.

Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH₄; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH₄.

The guanidine group of argirύne residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitization, for example, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using A- chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2- chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acid contemplated herein is shown in Table 4.

Crosslinkers can be used, for example, to stabilize 3D conformations, using homo- bifunctional crosslinkers such as the bifunctional imido esters having (CH₂)_n spacer groups with n=l to n-6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COOH). In addition, peptides can be conformationally constrained by, for example, incorporation of C_α and N _α-methylamino acids and the introduction of double bonds between C₀, and Cβ atoms of amino acids.

c-Myb alone is not considered a target encompassed by the present invention as it pertains to modulation of platelet levels, although a c-Myb antagonist may be used in combination with another c-Myb/p300 antagonist or in conjunction with other therapies. Hence, combination therapy wherein at least one therapeutic agent targets the c-Myb/p300 complex or a component therein is contemplated by the present invention, hi this case, c- Myb may be a target. Other agents or actions contemplated include platelet transfusion and the administration of a cytokine such as TPO or IL-I l. Accordingly, the present specification discloses a c-Myb/p300 antagonist for use in the treatment of conditions resulting in low platelet count in a subject, hi some embodiments, the antagonist or inhibitor is a small molecule, antibody, aptamer, peptide, hydrocarbon stapled peptide, genetic molecule or other membrane crossing agent that disrupts p300 KIX binding to c- Myb such as but not limited to 2-naphthol-AS-E-phosphate (KG-501) or pamoic acid (KG- 122) that binds to the KIX domain, or adjacent domains, including helices cd and o3 thereof as set out in Best et al, PNAS. 101: 17622-17627, 2004. hi a particular embodiment, the agent is a stapled peptide that disrupts p300 KIX binding to c-myb.

The present invention further contemplates methods of screening for cM/P antagonists by, for example, contacting a candidate drug with the cM/P complex or a component therein. The screening procedure includes an assay for the presence of binding between the drug and cM/P target as well as screening for any change in function of the complex or the ability to form the complex. Cell-based screening procedures are, therefore, also contemplated by the present invention.

In one aspect, the invention provides a method of identifying a candidate agent that enhances megakaryocytopoiesis and/or haematopoiesis, said method comprising: i) contacting the candidate agent with a system comprising a c-Myb/p300 complex or a component thereof, or a genetic sequence capable of producing same; and ii)determining the presence of a complex between the agent and the complex or a component thereof or a genetic sequence capable of producing same, a change in activity of the complex or a component thereof or a genetic sequence capable of producing same, or a change in the level of an indicator of the activity of the complex or a component thereof or a genetic sequence capable of producing same.

One form of assay involves competitive binding assays. In such competitive binding assays, p300 is labeled and incubated in the presence of a potential drug candidate. The amount of free (i.e. uncomplexed) label is a measure of the binding of the agent being tested against p300 or the inability for a complex to form. One may also measure the amount of bound, rather than free, p300. It is also possible to label the putative agent rather than a component of the c-Myb/p300 complex and to measure the amount of agent binding to the complex in the presence and in the absence of the drug being tested.

In some embodiments, agents that interact with (e.g. bind to) the c-Myb/p300 complex or a component thereof may be identified in a cell-based assay where a population of cells expressing the complex is contacted with a candidate agent and the ability of the candidate agent to interact with the complex is determined. Preferably, the ability of a candidate agent to interact with the c-Myb/p300 complex or a component thereof is compared to a reference range or control. In some embodiments, the ability of the candidate agent to interfere with p300-cMyb binding is determined in vitro. In another embodiment, a first and second population of cells expressing a c-Myb/p300 complex are contacted with a candidate agent or a control agent and the ability of the candidate agent to interact with the c-Myb/p300 complex or a component thereof is determined by comparing the difference in interaction between the candidate agent and control agent. If desired, this type of assay may be used to screen a plurality (e.g. a library) of candidate agents using a plurality of cell populations expressing a c-Myb/p300 complex. If desired, this assay may be used to screen a plurality (e.g. a library) of candidate agents. The cell, for example, can be of prokaryotic origin (e.g. E. colϊ) or eukaryotic origin (e.g. yeast or mammalian). Further, the cells can express the c-Myb/p300 complex endogenously or be genetically engineered to express the polypeptide. In some embodiments, the ability of a candidate agent to modulate c-Myb/p300 dependent gene transcription is assayed in cells transfected with a reporter construct using techniques known in the art.

In some embodiments, the invention comprises a method of identifying a candidate agent. In some embodiments, the agent enhances megakaryocytopoiesis and/or haematopoiesis. In other embodiments, the agent can be used for the treatment or amelioration of thrombocytopenia. In still other embodiments, the agent can be used to enhance the life span of platelets or other cellular blood products in vitro or ex vivo. In some embodiments, the method comprises: i) contacting the candidate agent with a system comprising a c- Myb/p300 complex or a component thereof, or a genetic sequence capable of producing same; and ii) determining the presence of a complex between the agent and the complex or a component thereof or a genetic sequence capable of producing same, a change in activity of the complex or a component thereof or a genetic sequence capable of producing same, or a change in the level of an indicator of the activity of the complex or a component thereof or a genetic sequence capable of producing same. Systems may be whole animal, whole cell or cell lysate systems as well as subcellular systems.

In some embodiments, potential candidate agents are inhibitors screened for their ability to bind to the p300 KIX domain or adjacent regions.

In some embodiments, KIX-binding drug candidates are tested for their ability to disrupt p300 KIX binding to c-Myb and/or their ability to disrupt c-Myb-dependent gene transcription in cells transfected with reporter constructs (such methods and kits known in the art for conducting same are described for example in Best et al., 2004 supra incorporated herein by reference).

Cell based screening assays include examining whether or not the drug can modify the symptoms of thrombocytopenic conditions or induce megakaryocytopoiesis or platelet production.

As indicated above, useful screening animal models include mice with or without the ability to produce TPO or its receptor MpI (e.g. MpT'^' or Mpl^+/+ mice), mice in an MpT^1' or Mpl^+/+ background and which carry a Plt6 mutation (in the gene encoding p300) and mice in an MpT*^' or Mpl^+/+ background which carry a PU4 mutation (in the gene encoding c- Myb) such mice are referred to herein, for example, as MpT'^' pSOO ^p"^6/+, MpT^ p 300 ^Pll6/Plt\ MpT'- c-Myb^PM/+ and MpT^A c-Myb^Plt4/PM or Mpt^/+ p300 ^p"^6/+, Mpl^+/+ p300 ^Plt6/Pm, Mpl^+/+ c-Myb^PM/+ and Mpl^+/+ c-Myb^plt4/PM . Multiple mutations such as heterozygous or homozygous Plt4 and PU6 maturation in an MpT^A or Mpl^+/+ animal are also contemplated herein.

Hence, another aspect of the present invention contemplates an animal model comprising a genetically modified animal having a genetic background selected from the list comprising

'U6/PH6

MpT'- p30(f^lt6/+, MpT¹- p30(f^lt6/Ph6, MpT^A p30(f^lt6/+ c-Myb^PU4/+ and MpT^Λ p30(f^l C-

Myb^PU4/PM Mpl^+/+ p30(f^m/+, Mpl^+/+ p30(f^m/PM, Mpt^/+ p30(f^!'^6/+ c-Myb^P"^4/+ and Mpl^+/+ _p3OO ^Pit6/pit6 _c__Myb ^PM/Pit\ _and c-Myb^plt4/Ph4 or offspring or back crosses thereof.

Conveniently, the animal is a rodent, such as a mouse.

Such mice may be screened to identify further targets within the network of molecule controlling hematopoiesis and megakaryocytopoiesis and platelet levels in a subject.

A c-Myb or p300 (or other component of the c-Myb/p300 complex) nucleic acid encoding a c-Myb or p300 polypeptide (or other polypeptide component of the c-Myb/p300 complex), including homologies and orthologues from species other than human, may be obtained by a process which comprises the steps of screening an appropriate library under stringent hybridization conditions with a labelled probe having the sequence of a desired nucleic acid and isolating full-length cDNA and genomic clones containing said nucleic acid sequence. Such hybridization techniques are well known in the art. One example of stringent hybridization conditions is where attempted hybridization is carried out at a temperature of from about 35° C to about 65° C using a salt solution of about 0.9M. However, the skilled person will be able to vary such conditions as appropriate in order to take into account variables such as probe length, base composition, type of ions present, etc. For a high degree of selectivity, relatively stringent conditions such as low salt or high temperature conditions, are used to form the duplexes. Highly stringent conditions include hybridization to filter-bound DNA in 0.5M NaHP04, 7% sodium dodecyl sulphate (SDS), ImM EDTA at 65° C, and washing in O.lxSSC/0.1% SDS at 68° C (Ausubel F.M. et al, eds., Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3, 1989). For some applications, less stringent conditions for duplex formation are required. Moderately stringent conditions include washing in 0.2xSSC/0.1% SDS at ' 42° C (Ausubel et al, 1989, supra). Hybridization conditions can also be rendered more stringent by the addition of increasing amounts of formamide, to destabilise the hybrid duplex. Thus, particular hybridization conditions can be readily manipulated, and will generally be chosen as appropriate. In general, convenient hybridization temperatures in the presence of 50% formamide are: 42° C for a probe which is 95-100% identical to the fragment of a gene encoding a polypeptide as defined herein, 37° C for 90-95% identity and 32° C for 70-90% identity.

One skilled in the art will understand that, in many cases, an isolated cDNA sequence will be incomplete, in that the region coding for the polypeptide is cut short at the 5' end of the cDNA. Methods to obtain full length cDNAs or to extend short cDNAs are well known in the art, for example RACE (Rapid amplification of cDNA ends; e.g. Frohman et al., Proc. Natl. Acad. Sci USA 85: 8998-9002, 1988). Recent modifications of the technique, exemplified by the Marathon® technology (Clontech Laboratories Inc.) have significantly simplified the search for longer cDNAs. This technology uses cDNAs prepared from mRNA extracted from a chosen tissue followed by the ligation of an adaptor sequence onto each end. PCR is then carried out to amplify the missing 5'-end of the cDNA using a combination of gene specific and adaptor specific oligonucleotide primers. The PCR reaction is then repeated using nested primers which have been designed to anneal with the amplified product, typically an adaptor specific primer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5' in the known gene sequence. The products of this reaction can then be analyzed by DNA sequencing and a full length cDNA constructed either by joining the product directly to the existing cDNA to give a complete sequence, or carrying out a separate full length PCR using the new sequence information for the design of the 5' primer.

Recombinant c-Myb and p300 polypeptides may be prepared by processes well known in the art from genetically engineered host cells comprising expression systems. The sequences of human p300 can be found under Genbank Accession Nos. NM_001429 for nucleotide and NP OO 1420 for corresponding protein sequence, respectively. The mouse p300 nucleotide sequence can be found as NM_177821 and NP_808489 for the corresponding protein sequence. The sequences of human c-Myb can be found under Genbank Accession Nos. NM 005375 for nucleotide and NP 005366 for corresponding protein sequence, respectively. The mouse c-Myb nucleotide sequence can be found as NM O 10848 and as NP_034978 for the corresponding protein sequence. These sequences and their accession numbers may be updated from time to time and all such modifications are contemplated and encompassed herein. The appropriate nucleic acid sequence may be inserted into an expression system by any variety of well known and routine techniques, such as those set forth in Sambrook et al., (Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbour laboratory Press, Cold Spring Harbour, NY, 1989). In some embodiments, c-Myb and p300 polypeptides or their encoding sequences may be generated synthetically.

If a polypeptide is to be expressed for use in cell-based screening assays, the appropriate nuclear targeting signal should be incorporated (see for a review, Pouton et al., Adv. Drug Delivery. Reviews, 59: 698-717, 2007). If the polypeptide is secreted into the medium, the medium can be recovered in order to isolate said polypeptide.

Polypeptides can be recovered and purified from recombinant cell cultures or from other biological sources by well known methods including, ammonium sulphate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, affinity chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, molecular sieving chromatography, centrifugation methods, electrophoresis methods, lectin chromatography, FPLC and HPLC. Combinations of these methods can be used as known by those skilled in the art.

The present invention is further directed to compositions such as pharmaceutical compositions comprising the c-Myb/p300 antagonist herein contemplated.

The terms "antagonist", "modifier", "compound", "active agent", "pharmacologically active agent", "medicament", "active" and "drug" are used interchangeably herein to refer to a molecule that induces a desired pharmacological and/or physiological effect and in particular antagonizes c-Myb/p300 activity or function or formation. The terms also encompass pharmaceutically acceptable and pharmacologically active ingredients of those active agents contemplated herein including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs and the like. When the terms "antagonist", "modifier", "compound", "active agent", "pharmacologically active agent", "medicament", "active" and "drug" are used, then it is to be understood that this includes the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs, etc. The term "compound" is not to be construed narrowly as it extends to inorganic and organic molecules including genetic molecules, peptides, polypeptides and proteins and chemical analogs thereof. The agents identified or screened in accordance with the present invention are proposed to be useful treating thrombocytopenia or conditions or disorder causing thrombocytopenia and to facilitate megakaryocytopoiesis and hemopoiesis. Reference to "an antagonist", "compound", "active agent", "pharmacologically active agent", "medicament", "active" and "drug" includes combinations of two or more actives such as one or more inhibitors of c-Myb/p300 activity or complex formation or a compound therein. A "combination" also includes a two-part or more such as a multi-part pharmaceutical composition where the agents are provided separately and given or dispensed separately or admixed together prior to dispensation.

The terms "effective amount" and "therapeutically effective amount" of an agent as used herein mean a sufficient amount of the agent to provide the desired therapeutic or physiological effect. Undesirable effects, e.g. side effects, are sometimes manifested along with the desired therapeutic effect; hence, a practitioner balances the potential benefits against the potential risks in determining what is an appropriate "effective amount". The exact amount required will vary from subject to subject, depending on the species, age and general condition of the subject, mode of administration and the like. Thus, it may not be possible to specify an exact "effective amount". However, an appropriate "effective amount" in any individual case may be determined by one of ordinary skill in the art using only routine experimentation. In some embodiments, an effective amount for a human subject lies in the range of about O.lng/kg body weight/dose to lg/kg body weight/dose. In some embodiments, the range is about lμ to Ig, about lmg to Ig, lmg to 500mg, lmg to 250mg, lmg to 50mg, or lμ to lmg/kg body weight/dose. Dosage regimes are adjusted to suit the exigencies of the situation and may be adjusted to produce the optimum therapeutic dose. For example, several doses may be provided daily, weekly, monthly or other appropriate time intervals. Thus, the time and conditions sufficient for promotion of platelet production can be determined by one skilled such as a medical practitioner who is able to specify a therapeutically effective amount.

By "pharmaceutically acceptable" carrier, excipient or diluent is meant a pharmaceutical vehicle comprised of a material that is not biologically or otherwise undesirable, i.e. the material may be administered to a subject along with the selected active agent without causing any or a substantial adverse reaction. Carriers may include excipients and other additives such as diluents, detergents, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives, and the like.

Similarly, a "pharmacologically acceptable" salt, ester, emide, prodrug or derivative of a compound as provided herein is a salt, ester, amide, prodrug or derivative that this not biologically or otherwise undesirable.

Hence, the present invention provides a pharmaceutical composition comprising a c- Myb/p300 antagonist and one or more pharmaceutically acceptable carriers, diluents and/or excipients.

The present invention further contemplates a method for the treatment or prophylaxis of a subject with thrombocytopenia in a subject, the method comprising administering to the subject an amount of an agent effective to inhibit formation, expression or activity of the transcriptional regulatory complex c-Myb/p300 for a time and under conditions sufficient for promotion of platelet production.

Another aspect of the present invention provides a method for promoting megakaryocytopoiesis and/or hemopoiesis in a subject, the method comprising administering to the subject an effective amount of an agent which inhibits the formation, expression or activity of the transcription regulatory complex c-Myb/p300 for a time and under conditions sufficient for promotion of platelet production.

Accordingly, the present invention also provides an agent which inhibits or down regulates the activity or formation of c-Myb/p300 for use in promoting megakaryocytopoiesis and/or haematopoiesis in a mammalian subject.

In some embodiments the subject has or is likely to have thrombocytopenia. Thrombocytopenia may be as a result of a genetically inherited mutation, spontaneous mutation, or as a result of cytotoxic treatment, such as cancer therapy, or as a result of surgery, such as bone marrow transplantation. Thrombocytopenia may also be as a result of chronic liver disease and viral infections, including HIV/ AIDS, an autoimmune disease or haematological disease.

Thus, in some embodiments the invention provides a c-Myb/p300 antagonist for use in the treatment of thrombocytopenia. In some embodiments, the antagonist is a small molecule, aptamer, inhibitory RNA, peptide, peptidemimetic or constrained peptide, stapled peptide, antibody or antigen binding agent.

A medical protocol is also provided comprising the use of an agent which inhibits the activity or formation of c-Myb/p300 and optionally combining this with platelet transfusion or another procedure such as the administration of a c-Myb antagonist and/or TPO and/or IL-I l.

Hence, provided herein is a combination therapeutic protocol for the treatment or prophylaxis of thrombocytopenia, the protocol comprising the administration of a medicament as defined herein and one or more other treatments such as platelet transfusion, a c-Myb antagonist and/or TPO and/or IL-11.

The present invention is further described by the following non-limiting Examples, hi these Examples mice were generated and haematology conducted as described below:

Plt6 mice. The founder Plt6 mouse was identified among G₁ offspring of male Mpl'^~ C57BL/6 mice that were treated with ENU as described (Carpinelli et al., Proc Natl Acad Sci USA 101: 6553-6558, 2004). For mapping, affected heterozygous PH6/+ Mpl'^~ mice on a C57BL/6 background were crossed to Mpl^+/~ mice on a 129Sv background. PU6/+ MpT^1' Fi animals were then intercrossed to produce 373 mice in the F₂ generation. Platelet counts were used to identify the F₂ mice as +/+, PH6/+ or Plt6/Plt6 and DNA was prepared from each mouse according to described methods (Carpinelli et al., 2004 supra). 148 simple sequence length polymorphisms (SSLP) spaced evenly throughout the genome were amplified and analysed, essentially as described (Dietrich et al., Genetics 131: 423- 447, 1992). PU6 was found to reside on chromosome 15 and the location was refined via analysis of additional markers in this region. Each of the exons and intron-exon boundaries of the p300 gene was amplified by PCR and sequenced on an ABI automatic sequencer according to the manufacturer's instructions. The presence of the mutation exclusively in Plt6 mutants was confirmed in each of 3 PU6/PU6, PU6/+ and +/+ mice.

Haematology. Peripheral blood counts were determined using automated (Advia 120, Bayer) techniques and clonal agar cultures of bone marrow cells or spleen cells, stimulated with 100 ng/ml murine SCF, 10 ng/ml murine IL-3 and 4 U/ml human EPO, were performed as previously described (Alexander et al., Embo J 14: 5569-5578, 1995). Megakaryocyte counts were performed from sections of sternum and spleen following staining with haematoxylin and eosin. CFU-s were enumerated by intravenous injection of bone marrow cells into recipient mice that had been irradiated with 11 Gy of γ-irradiation. Spleens were removed after 8 or 12 days, fixed in 60% v/v ethanol/30% v/v chloroform/ 10% v/v acetic acid, and the numbers of macroscopic colonies were counted.

EXAMPLE 1

Suppression of thrombocytopenia in Mpt'^~ mice

A mutagenesis screen was conducted for suppressors of Mpϊ ^' thrombocytopenia, and a founder PH6 mouse was identified among the first generation offspring of an ENU-treated Mpϊ'^' mouse as an outlier with a platelet count of 371 x 10⁶/ml, compared with 105 ± 44 xlO⁶/ml in littermate mice. Approximately half the offspring of matings between affected Plt6 mice and untreated Mpϊ'^' mice exhibited elevated platelet counts consistent with inheritance of a suppressor of thrombocytopenia. In offspring from inter-crosses between PH6/+ parents, mice displaying platelet counts typical of PH6/+ mice were observed along with occasional mice displaying supraphysiological platelet counts. It was subsequently established that PU6/PU6 homozygotes died in utero or as neonates (of 71 mice weaned, ratio of Plt6/PH6:Plt6/+:+/+ = 0.04:2.0:1.0) with occasional survivors exhibiting very high platelet numbers (Table 3).

To map the chromosomal location of the Plt6 mutation, a (C57BL/6 x 129/Sv)F2 cohort of mice segregating the Plt6 mutation were generated as described above, bled and phenotypically categorized as having platelet counts typical of un-mutated Mpϊ'^', PH6/+ or PU6/PU6 mice and then genotyped using simple sequence length polymorphisms (SSLP) spaced evenly throughout the genome. Markers found to be homozygous 129/Sv are shown in white, heterozygous in gray and homozygous C57BL/6 in black (Figure IA).

The mutation was localized to a region of approximately 1.2Mb between D15AahA10 and D15AahA3 (Figure IA). This region of chromosome 15 included the gene encoding p300. The DNA coding regions of p300 in DNA from PU6/PU6, Plt6/+ and +/+ mice were sequenced and a single T to A mutation identified resulting in substitution of tyrosine (Y) for asparagine (N) at residue 630 within the KIX domain (Figures IB and C).

Cohorts of Mpϊ'^' p30(f^m/PM, Mpϊ'^' p300^p"^6/+ and Mpϊ'^' p300^+/+ mice were established for phenotypic characterization. In Mpϊ'^' p30(f"⁶'⁺ mice, suppression of thrombocytopenia was partial, with platelet counts elevated up to three- fold relative to Mpϊ'^' p300^+/+ controls and reaching levels approximately half that of MpI^{+ +} p300^{+ +} mice. Despite the absence of a functional TPO/Mpl system, thrombocytopenia was not only suppressed in MpT'^' _p3OOP^it6/pit6 _{mice^ but platelet counts} reached levels up to 20-fold that of MpT'' p300^+/+ mice and twice that of wild-type mice (Table 3). The haematocrit, and numbers of red and white blood cells were unaltered in MpT'^' p30(f^lt6/+ and MpT'^' p30(f^lt6/plt6 mice (Table 3).

Significant increases in the numbers of megakaryocytes and megakaryocyte progenitor cells in the bone marrow and spleens accompanied the suppression of thrombocytopenia in MpT'^' p30(f"^6/+ mutants and these changes were more profound in MpT'^' p300^pll6/p!t6 mice (Figure 2). The total number of myeloid progenitor cells was significantly elevated in the spleens oiPltό homozygote and heterozygote mice (MpT'^' p300^plt6/plt6: 25±2± colonies per 10⁵ cells (n=3), MpT'^' p30(f^h6/+: 11±9± (n=7) and MpT'^' p300^+/+: 3±3 (n=9)) while in the bone marrow excess progenitor cells were evident in homozygotes, but not heterozygotes (MpT'^' p30(f^lt6/Plt6: 68±33 colonies per 2.5xlO⁴ cells (n=3), MpT'^' p30(f"⁶'⁺: 34±17 (n=7)± 9 and MpT'^' p300^+/+: 22±11 (n=9)±2). Consistent with a general increase in haematopoietic progenitor cells in Pltό mutant mice, elevated numbers of CFU-s were also apparent in the bone marrow of MpT'^' p30(f^lt6'^plt6 and MpT'^' p30(f^lt6/+ mice (Figure 2).

The data herein establish a key role for the c-Myb/p300 transcriptional regulatory complex in restraining megakaryocytopoiesis. Moreover, the data establish that inhibition of c- Myb/p300 results in very significant production of megakaryocytes and platelets in the absence of TPO signaling. Thus, the c-Myb/p300 mutant mice indicate that pharmaceutical inhibition of the Myb/p300 complex is useful in treating thrombocytopenia.

EXAMPLE 2

Response ofPlt4/+ and PU6/+ mice to 5-fluorouracil (5-FU) and bone marrow (BM) transplantation

Mice lacking c-Mpl, the receptor for the platelet-stimulating cytokine thrombopoietin (TPO), are thrombocytopenic, with platelet counts less than 25% that of normal mice (Alexander et al, Blood 57:2162-2170, 1996). ENU-induced mutations in the genes encoding c-Myb (Plt3 and PU4) or p300 (Plt6) suppress this thrombocytopenia, with MpT^f~ c-Myb ^plt4/+ and MpT^f~ p300 ^Plt6/+ mice showing platelet counts ~3-4-fold higher than MpF^A controls (see Example 1) [Carpinelli et al., 2004 supra]. Mpϊ'^~ c-Myb ^PM/PM and Mpf^' p300 ^plt6/plt6 mice, in which the mutations are homozygous, have supraphysiological platelet production, with platelet counts ~3-5-fold higher than wild-type mice (see Example 1) [Carpinelli et al, 2004 supra}. These data indicate that pharmacological inhibition of c-Myb and/or p300 is useful in the treatment of thrombocytopenia.

To more directly investigate whether inhibition of c-Myb or p300 activity is beneficial in ameliorating thrombocytopenia associated with cancer therapy, recovery of blood cells was investigated in c-Myb ^PM/+ and p300 ^p"^6/+ mice treated with the cytotoxic drug 5- fluorouracil (5-FU) or following bone marrow (BM) transplantation. Heterozygous mutant mice on a wild-type rather than MpT^1' genetic background were used to better model humans and in acknowledgment that pharmacological agents may not fully inhibit protein action.

Initially, haematopoiesis was characterized in un-manipulated c-Myb ^PM/+ and p300 ^p"^6/+ mice. Peripheral blood was collected into EDTA tubes for subsequent automated analysis on an Advia 120 haemato logical analyser. Platelet counts in these mice were indistinguishable from wild-type controls, as were the numbers of red blood cells and morpho logically recognisable subsets of white blood cells (Table 1). Numbers of megakaryocytes, the precursor cells from which platelets are produced, were scored by enumeration of morpho logically recognizable megakaryocytes via light microscopic assessment of histological sections of bone marrow and spleen as previously described (Alexander et al., 1996 supra) and were also present in normal numbers in c-Myb ^p'^{4 +} and p300 ^Plt6/+ mice (Table 1). Counts of haematopoietic progenitor cells [by clonal assay of bone marrow and spleen cells in vitro using cytokines to stimulate colony formation as previously described (Alexander et al., 1996 supra)] revealed normal numbers of progenitor cells for megakaryocytes and other blood cell lineages (Table 2). Thus, unlike mice on a MpT'^' genetic background, on a wild-type background the c-Myb ^PM and p300 ^PM mutations do not significantly enhance megakaryocyte and platelet production when heterozygous.

Cohorts of mice were injected intra-peritoneally with 150mg/kg of 5-FU and then peripheral blood cell counts measured (as described above) in groups of mice at various times post-treatment, hi c-Myb ^PM/+ mice treated with 5-FU, the cytotoxic effects of the drug caused a reduction in platelet numbers followed by recovery to pre-treatment levels with kinetics identical to wild-type controls. However, while the excess platelet production that characterizes responses to 5-FU peaked at days 11-14 post-treatment in wild-type mice, excessive platelet production was observed in c-Myb ^p!t4/+ mice for an additional 7- 10 days and reached a peak value 1.6-fold higher than controls (Figure 3). Recovery of white blood cells and red blood cells (haematocrit) was similar in c-Myb ^PM/+ mice to wild- type mice, with mildly delayed recovery of white blood cells in the c-Myb ^PM/+ animals (Figure 3). Similar observations were made in p300 ^plt6/+ mice recovering from 5-FU treatment: significantly prolonged production of platelets at excess levels was observed relative to wild-type controls, although the magnitude and duration was not as great as in c-Myb ^plt4/+ mice (Figure 4). No significant differences in recovery of white blood cells and red blood cells (haematocrit) was evident in p300 ^pll6/+ mice relative to controls (Figure 4).

Wild-type control mice receiving a bone marrow transplant (full body irradiation in two equal doses of 5.5Gy several hours apart followed by intravenous injection of 2xlO⁵ syngeneic bone marrow cells) displayed severe thrombocytopenia with a platelet nadir at day 11, followed by gradual recovery of circulating platelet numbers. c-Myb ^PM/+ transplant recipients also developed thrombocytopenia with similar kinetics, although the depth of the platelet nadir was marginally, but significantly, less severe than control mice, and the rate of platelet recovery was significantly enhanced (Figure 5). No significant differences in recovery of white blood cells and red blood cells (haematocrit) were evident in c-Myb ^PM/+ mice relative to controls (Figure 5). Similar observations were made in p300 ^Plt6/+ mice recovering from bone marrow transplantation. As observed in platelet recovery from 5-FU treatment, while the rate of platelet recovery in p300 ^p"^6/+ mice was greater than wild-type controls, it was not as significant as in c-Myb ^PM/+ mice (Figure 6). No significant differences in recovery of white blood cells and red blood cells (haematocrit) was observed in p300 ^plt6/+ mice relative to controls (Figure 6).

Together, these data show that platelet production during recovery from thrombocytopenia induced by 5-FU or bone marrow transplantation is enhanced in c-Myb ^PM/+ and/? 300 ^Ph6/+ mice relative to wild-type animals, showing that pharmacological inhibition of c-Myb and/or p300 is useful in treating thrombocytopenia associated with treatments such as but not limited to cytotoxic treatments or bone marrow transplantation.

TABLEl Haematological profile ofPlt4/+ andPlt6/+ mutant mice

Genotype

+/+ PU4/+ PU6/+

Peripheral Blood (Advia) n=13 n=9 n=16

Platelet Count (xlO-⁶/ml) 1093 ±133 1157± 167 1166 ±185

Red Cell Count (xlθ^"6/ml) 10.9 ±0.4 10.9 ±0.3 10.8 ±0.4

Haematocrit (%) 49.6 ±1.4 49.3 ±1.2 50.2 ±1.4

White Cell Count (xlO-⁶/ml) 9.9 ±1.8 6.9 ±2.5 8.8 ±2.1

Neutrophils 0.90 ±0.20 0.64 ± 0.26 0.93 ±0.18

Lymphocytes 8.4 ±1.6 5.8 ±2.1 7.2 ±1.9

Monocytes 0.12 ±0.05 0.08 ± 0.04 0.11 ±0.03

Eosinophils 0.19 ±0.05 0.18 ±0.07 0.23 ±0.11

Bone Marrow n=4 n=4 n=4

Megakaryocytes (per 10 hpfp 77 ±9 87 ±18 73 ±12

Spleen n=4 n=4 n=4

Megakaryocytes (per 10 hpf)# 20 ±7 24 ±18 49 ±18

Means ± standard deviations are shown. # determined from histological sections, hpf, high powered field (x600 for bone marrow, x200 for spleen).

TABLE 2

Haematopoietic progenitor cells in PU4/+ and Plt6/+ mutant mice

Genotype Number of Colonies per 25,000 BM cells

SCF + IL-3 + EPO

Blast G GM M Eo Meg

+/+ 1166±±77 1166±±77 1199±±99 18 ±7 0.5 ±0.5 22 ±13

PU4/+ l1O0±±l1 1111 ±±44 1166±±44 20 ±6 0 28 ±4

PU6/+ 1122 ±±77 1166±±88 2200 ±±1100 20 ±12 0 31 ±20

Number of Colonies per 100,000 spleen cells

SCF + IL-3 + EPO

Blast G GM M Eo Meg

+/+ 5±3 2±1 2± 1 2± 1 0 12±2

PU4/+ l±l l± 1 l±l l± 1 0 15 ± 8 PU6/+ 3±3 l±l 2±1 l± 1 0 19 ±6

Means ± standard deviations are shown. n=3-4 mice per genotype

EXAMPLE 3

Supra-physiological platelet production in normal mice

Enhancement of megakaryocytes and megakaryocyte progenitor cells within the bone marrow and spleen

To determine the effects of the p30(f^m mutation on a wild-type genetic background, the mutant allele was also bred on a Mpt^/+ genetic background.

Mice carrying the PU6 mutation on a wild-type genetic background were generated by crossing Mp^ p30(f^lt6/+, and then subsequently Mpl^+Λ p30(f^h6/+rmcQ, with Mpl^+/+

C57BL/6 wild-type mice followed by intercrossing Mpl^+/+ p30(f^lt6/+ mice. Bone marrow transplantation was performed by intravenous injection of 5x10⁶ bone marrow cells from

Ly5.2 Mpl^+/+ p300^p!t6/pltδ or control Ly5.2 Mpl^+/+ p300^+/+ mice into wild-type Ly5.1 recipients. Several hours prior to transplantation, the recipient mice were irradiated with HGy of γ-irradiation in two equal doses given several hours apart from a ¹³⁷Cs source

(Atomic Energy, Ottawa, Canada). Transplanted mice were maintained on oral antibiotic

(l.lg/L neomycin sulfate; Sigma, St. Louis, MO) and analysed 3 months post-engraftment.

As observed on a MpT'^' background, the majority of Mpl^+/+ p30(f"^6/p"⁶ mice failed to survive to weaning (Plt6/Plt6:Plt6/+;+/+ = 0.2:2.0:1.3, n=81). However, in Mpl^+/+ p300^Pll6/PI'⁶ survivors, supraphysiological platelet counts were present, and lymphopenia was also evident (Table 3).

To overcome the paucity of adult MpI^{+ +} p30(f ^t6/plt6 mice available for phenotypic analysis, cohorts of wild-type mice were irradiated and transplanted with bone marrow cells from each of two Mpl^+/+ p30(f^lt6/plt6 and two control Mpl^+/+ p300^+/+ mice. Analysis of peripheral blood and haematopoietic organs, taking advantage of differences in the congenic Ly5 cell surface marker between donor and recipient mice, confirmed successful engraftment, with over 80% contribution of donor haematopoiesis in all mice analysed. The thrombocytosis and lymphopenia characteristic of the donor Mpl^+/+ p30(f^lt6/pm mice was present in the recipients of bone marrow of this genotype (Table 3), establishing that the Plt6 phenotype is bone marrow instrinsic. Consistent with the data from Pltό mutants on a Mpl^A background, the thrombocytosis in recipients of Mpl^+/+ p30(f^h6/plt6 marrow was accompanied by a significant increase in the numbers of megakaryocytes and megakaryocyte progenitor cells in the bone marrow and spleen (Figure 8). The total number of myeloid progenitor cells was also significantly elevated in the bone marrow (Mpl^+/+ p30(f"^6/Pl'⁶: 129±8 colonies per 2.5xlO⁴ cells, (n=3); Mpl^+/+ _P300^+/+: 93± 23, (n=4)) and spleen (Mpl^+/+ p30(f^m/pm: 79±18 colonies per 10⁵ cells, (n=4); Mpl^+/+ p300^+/+: 19± 7, (n=4)).

To further characterise the lymphopenia associated with homozygous PU6 mutation on a Mpl^+/+ genetic background, flow cytometric analysis was performed on tissues from two Mpl^+/+ p30(f"^6/Plt6 mice (data not shown) and the cohort of Mpl^+/+ p30(f^lt6/PU6 transplant recipients (Figure 9). The relative numbers of T cells were not significantly altered, rather the lymphopenia in Plt6 mutants resulted from reduced numbers of B-cells. This was accompanied by significant reductions in B cells in the bone marrow, spleen and lymph nodes (Figure 9). In the peripheral lymphoid tissues, reductions in IgM⁺ B cells were also observed. Consistent with the flow cytometry data, histological examination of spleens revealed smaller than normal lymphoid follicles in both Mpl^+/+ p30(f^h6/plt6 mice examined and in the majority of the recipients of Mpl^+/+ p300^p"^6/p"⁶ bone marrow. There were no significant alterations in the relative numbers of T cells in the thymus, spleen or lymph nodes in Mpl^+/+ p30(f^lt6/Plt6 mice or the recipients of Mpt^/+ _P30(f"^6/p"⁶ bone marrow relative to Mpl^+/+ p300^+/+ controls, as assessed via CD4 and CD8 antibody staining (data not shown).

Previous analyses of mice with mutations in p300 or c-Myb have described anemia and alterations in erythroid precursor cells within haematopoietic tissues (Carpinelli et al, 2004 supra; Kasper et al, 2002 supra; Sandberg et al, Dev Cell. 8: 153-166, 2005). Anemia was not evident in the small cohorts of p30(f^lt6/plt6 mice examined, on either a Mpt^/+ or MpT^1' genetic background and although a trend toward reduced red blood cell number and haematocrit in the recipients of Mpl^+/+ p30(f^ll6/plt6 bone marrow relative to control mice was observed, this was not statistically significant (Table 3). However, an increased proportion of Terl l9⁺ cells was observed in the spleens of MpI^{+ +} p300f ^{l t6} bone marrow recipients via flow cytometry (recipients of Mpl^+/+ p30(f"^6/p"⁶ bone marrow: 30±5% versus Mpt^/+ p300^+/+ recipients: 49±7%, n=4) and was confirmed by histological observations of increased numbers of nucleated erythroid cells in sections of spleens from Mpl^+/+ p30(f^h6/Plt6 bone marrow recipients compared with recipients of normal marrow (data not shown).

Histological analysis of the major non-haematopoietic organs of mice bearing p300^p 7'((S mutations on either a Mpl^+/+ or Mpϊ'^~ genetic background, as well as of recipients of Mpl^+/+ p30(f"^6/Plt6 bone marrow, revealed no consistent changes compared with respective p300^+/+ controls.

TABLE 3. Peripheral blood counts in PIt6 mutant mice

Transplant Recipients

MpI^ MpI ^+/+ MpI ^+/+

PU6/PU6 PU6/+ +/+ PU6/PU6 +/+ PU6/PU6 +/+ n=5 n=9 n=8 n=5 n=9 n=7 n=8

Platelets (xlθ^"6/ml) 2732 ± 462* 541 ± 194* 208 ± 62 1954 ±171* 1066 ±142 1917 ±164* 1081 ±257

Red Cell Count (xlθ^"6/ml) 10.6 ±0.8 10.8 ±0.6 10.0 ±0.5 10.3 ±0.2 10.7 ±0.3 9.0 ±0.6 10.0 ±0.9

Haematocrit (%) 52.8 ±4.9 52.5 ±4.0 49.5 ±2.5 48.1 ±1.9 49.1 ±1.2 49.6 ±2.7 52.4 ±4.1

White cells (xlθ^"6/ml) 6.2 ±2.7 6.6 ±2.1 4.6 ±1.8 5.4 ±1.2* 10.3 ±1.4 6.2 ±1.9* 10.6 ±2.2

Neutrophils (xlθ^"6/ml) 0.48 ± 0.32 0.65 ± 0.44 0.47 ± 0.37 0.65 ±0.12 0.89 ±0.15 0.27 ± 0.20* 0.75 ±0.30

Lymphocytes (xlO^'Vml) 4.8 ±2.2 5.5 ±2.0 3.9 ±1.4 4.2 ±1.2* 8.7 ±1.3 5.3 ±1.6* 9.2 ±2.0

Monocytes (xlθ^"6/ml) 0.07 ± 0.07 0.07 ± 0.07 0.05 ± 0.04 0.06 ± 0.03 0.11 ±0.03 0.08 ±0.10 0.15±0.10

Eosinophils (xlO^'Vml) 0.06 ± 0.04 0.19 ±0.06* 0.10 ±0.04 0.08 ± 0.04* 0.20 ± 0.04 0.13±0.10 0.25 ± 0.20

* p<0.05 after adjustment 1 for multiple testing for comparison of data from PU6/PU6 or PU6/+ mice with that of +/+ mice in each of the Mpt'^~, Mpl^+/+ or Mpl^+/+ transplant recipie mt groups.

EXAMPLE 4

PU6 mutation in KIX domain ofp300 hinders interaction with c-Myb leading to increased megakaryocyte development and platelet production

A structural model of the p300 KIX domain, prepared by homology modeling based on the solution structure of the highly homologous CBP KIX domain (Radhakrishnan et al., Cell. 91: 741-752, 1997), indicated that the side chain of Y630 is a component of the KIX domain's hydrophobic core. Inspection of amino acid side chains in proximity (<4.5 Angstrom) to Tyr630 in this model identified several putative hydrophobic interactions that are likely to be disrupted by the Y630N mutation (blue side chains, Figure 1C) resulting in structural distortion.

The Plt6 suppression of thrombocytopenia phenotype shows remarkable similarity to that of Mpl'^~ mice with mutations in c-Myb (Carpinelli et al., 2004 supra), a p300 partner protein. Previous studies have established a role for the residues, Y650, A654 and Y658 on the α3 helix of CBP, and their homologs in murine p300 (Y631, A635 and Y639, drawn in green on Figure 1C), in mediating interaction with c-Myb (Kasper et al., 2002 supra). Thus, in accordance with one aspect of the present invention the p30(f"⁶ mutation disrupts p300-c-Myb binding. GST fusions of p300 KIX domains containing the wild-type or Y630N mutant sequence were expressed and purified from E. coli and their capacity to bind ³⁵S-labelled in vitro transcribed/translated c-Myb was examined in pulldown experiments.

The cDNAs encoding wild-type and Y630N murine p300 KIX domains (residues 567-667) were cloned into a modified pGEX-2T vector (GE Healthcare, Piscataway, NJ). GST and

GST-KIX fusions were expressed and purified from E. coli BL21-CodonPlus-RIL

(Stratagene, La Jolla, CA). Cultures were grown to an optical density of 0.6-0.8 at 37°C before induction with 0.5mM IPTG and incubation at 15°C for 16 hours. Harvested cells were lysed by sonication at 4°C in 20OmM NaCl, 2OmM HEPES pH7.5, 2mM dithiothreitol (buffer A) supplemented with ImM phenylmethylsulfonyl fluoride. The lysates were clarified by centrifugation and incubated with glutathione sepharose resin (GE Healthcare) for 1 hour at 4⁰C, before the beads were washed extensively with buffer A.

The cDNA encoding full-length murine c-Myb was cloned into the vector, pET15b (Novagen, San Diego, CA), under the T7 promoter. ³⁵S-labelled c-Myb was generated from the TNT in vitro transcription/translation reaction (Promega, Madison, WI) from 750ng DNA in a 50μL reaction volume, according to manufacturer's instructions.

Pulldown experiments were performed to examine the capacity of GST and GST-KIX proteins to bind c-Myb. An equal volume of beads bound to 2μg GST, GST-wild-type KIX or GST-Y630N KIX was mixed with 5μL ³⁵S-labelled in vitro transcribed/translated c-

Myb in a 30μL volume at 4°C for 40min. Beads were washed four times with 0.5mL buffer A supplemented with 0.5% v/v Nonidet P40 at 4°C, boiled with reducing SDS-

PAGE loading dye and resolved by SDS-PAGE. The gel was stained with Coomassie Blue to visualize bait levels before drying under vacuum and phosphor screen exposure.

Phosphorimages were read 16-48 hours later and densitometric analysis of bands performed using the software, Image Gauge 4.0 (Fujifϊlm, Tokyo, Japan).

The GST-KIX fusion protein containing the Y630N mutation bound to c-Myb with markedly reduced affinity: on average, a ~7-fold reduction in the amount of c-Myb retained by GST-KIX (Y630N) relative to GST-KIX (wild-type) was observed (Figure 7). These data demonstrate that the Y630N mutation in the KIX domain of p300 severely hinders c-Myb binding, but does not completely abrogate this interaction.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. TABLE 4 Codes for non-conventional amino acids

Non-conventional Code Non-conventional Code amino acid amino acid

α-aminobutyric acid Abu L-N-methylalanine Nmala α-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-Nmethylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile

D-alanine Dal L-N-methylleucine Nmleu

D-arginine Darg L-N-methyllysine Nmlys

D-aspartic acid Dasp L-N-methylmethionine Nmmet D-cysteine Dcys L-N-methylnorleucine Nmnle

D-glutamine DgIn L-N-methylnorvaline Nmnva

D-glutamic acid DgIu L-N-methylornithine Nmorn

D-histidine Dhis L-N-methylphenylalanine Nmphe

D-isoleucine DiIe L-N-methylproline Nmpro D-leucine Dleu L-N-methylserine Nmser

D-lysine Dlys L-N-methylthreonine Nmthr

D-methionine Dmet L-N-methyltryptophan Nmtrp

D-ornithine Dorn L-N-methyltyrosine Nmtyr

D-phenylalanine Dphe L-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine Nmetg

D-serine Dser L-N-methyl-t-butylglycine Nmtbug

D-threonine Dthr L-norleucine NIe

D-tryptophan Dtφ L-norvaline Nva

D-tyrosine Dtyr α-methyl-aminoisobutyrate Maib D- valine Dval α-methyl-γ-aminobutyrate Mgabu

D-α-methylalanine Dmala α-methylcyclohexylalanine Mchexa

D-α-methylarginine Dmarg α-methylcylcopentylalanine Mcpen

D-α-methylasparagine Dmasn α-methyl-α-napthylalanine Manap

D-α-methylaspartate Dmasp α-methylpenicillamine Mpen D-α-methylcysteine Dmcys N-(4-aminobutyl)glycine NgIu

D-α-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg

D-α-methylhistidine Dmhis N-(3-aminopropyl) glycine Norn

D-α-methylisoleucine Dmile N-amino-α-methylbutyrate Nmaabu

D-α-methylleucine Dmleu α-napthylalanine Anap D-α-methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionine Dmmet N-(2-carbamylethyl)glycine NgIn

D-α-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn

D-α-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine NgIu

D-α-methylproline Dmpro N-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycine Ncbut

D-α-methylthreonine Dmthr N-cycloheptylglycine Nchep

D-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex

D-α-methyltyrosine Dmty N-cyclodecylglycine Ncdec

D-α-methylvaline Dmval N-cylcododecylglycine Ncdod D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct

D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro

D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund

D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm

D-N-niethylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine Nbhe D-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine Narg

D-N-methylglutamate Dnmglu N-( 1 -hydroxyethyl) glycine Nthr

D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser

D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis

D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp D-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate Nmgabu N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet

D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen

N-methylglycine NaIa D-N-methylphenylalanine Dnmphe N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro N-(l-methylpropyl) glycine Nile D-N-methylserine Dnmser N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr

D-N-methyltryptophan Dnmtφ N-(l-methylethyl)glycine Nval

D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap

D-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr

L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys

L-ethylglycine Etg penicillamine Pen

L-homophenylalanine Hphe L-α-methylalanine Mala

L-α-methylarginine Marg L-α-methylasparagine Masn L-α-methylaspartate Masp L-α-methyl-t-butylglycine Mtbug

L-α-methylcysteine Mcys L-methylethylglycine Metg

L-α-methylglutamine MgIn L-α-methylglutamate MgIu

L-α-methylhistidine Mhis L-α-methylhomophenylalanine Mhphe

L-α-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet L-α-methylleucine Mleu L-α-methyllysine Mlys

L-α-methylmethionine Mmet L-α-methylnorleucine MnIe

L-α-methylnorvaline Mnva L-α-methylornithine Morn

L-α-methylphenylalanine Mphe L-α-methylproline Mpro

L-α-methylserine Mser L-α-methylthreonine Mthr L-α-methyltryptophan Mtφ L-α-methyltyrosine Mtyr L-α-methylvaline Mval L-N-methylhomophenylalanine Nmhphe N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycine carbamylmethyl)glycine 1 -carboxy- 1 -(2,2-diphenyl- Nmbc ethylamino)cyclopropane

TABLE 6

Exemplary and Preferred Amino Acid Substitutions

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Claims

CLAIMS:

1. Use of the c-Myb/p300 transcriptional regulatory complex or a component thereof, other than c-Myb alone, in the manufacture of a medicament in the treatment of thrombocytopenia in a subject.

2. Use of Claim 1 wherein the component of c-Myb/p300 used is p300 or a functional homolog thereof.

3. Use of an agent which inhibits the formation, expression or activity of the c-Myb/p300 transcriptional regulatory complex or a component thereof, other than c-Myb alone, in the manufacture of a medicament in the treatment of thrombocytopenia in a subject.

4. Use of claim 3 wherein the agent is a small molecule, an antibody, a nucleic acid or a stapled peptide.

5. The use of any one of Claims 1 to 4 wherein the subject is a human.

6. A method for the treatment or prophylaxis of a subject with thrombocytopenia or who is at risk of developing same said method comprising administering to the subject an amount of an agent effective to inhibit formation, expression or activity of the transcriptional regulatory complex c-Myb/p300, other than c-Myb alone, for a time and under conditions sufficient for promotion of platelet production.

7. The method of Claim 6 wherein the subject is a human.

8. Use of the c-Myb/p300 transcriptional regulatory complex or a component thereof, other than c-Myb alone, in the manufacture of a medicament to induce or otherwise facilitate megakaryocytopoiesis or haematopoiesis in a subject.

9. Use of the c-Myb/p300 transcriptional regulatory complex or a component thereof, other than c-Myb alone, in the manufacture of a medicament to induce or otherwise facilitate production of platelets in a subject.

10. Use of the c-Myb/p300 transcriptional regulatory complex or a component thereof, other than c-Myb alone, in the manufacture of a medicament to induce or otherwise facilitate treatment of conditions resulting in a low platelet count in a subject.

11. Use of Claim 8 or 9 or 10 wherein the component of c-Myb/p300 used is p300 or a functional homolog thereof.

12. Use of an agent which inhibits the formation, expression or activity of the c-Myb/p300 transcriptional regulatory complex or a component thereof, other than c-Myb alone, in the manufacture of a medicament to induce or otherwise facilitate megakaryocytopoiesis or haematopoiesis in a subject.

13. Use of an agent which inhibits the formation, expression or activity the c-Myb/p300 transcriptional regulatory complex or a component thereof, other than c-Myb alone, in the manufacture of a medicament to induce or otherwise facilitate production of platelets in a subject.

14. Use of an agent which inhibits the formation, expression or activity the c-Myb/p300 transcriptional regulatory complex or a component thereof, other than c-Myb alone, in the manufacture of a medicament to induce or otherwise facilitate treatment of conditions resulting in a low platelet count in a subject.

15. Use of any one of Claims 8 to 14 wherein the subject is a human.

16. A combination therapeutic protocol for the treatment or prophylaxis of thrombocytopenia said protocol comprising the administration of a medicament as defined in any one of Claims 1 to 5 or 8-15 and one or more other treatments.

17. The protocol of Claim 16 wherein the other treatment is a platelet transfusion.

18. The protocol of Claim 16 wherein the other treatment is the administration of a c-Myb antagonist.

19. The protocols of Claim 16 wherein the other treatment is the administration of thrombopoietin and/or IL-11.

20. An animal model comprising a genetically modified animal having a genetic background selected from the list comprising MpT'^' p30(f^lt6/+, MpT'^' p30(f^U6/plt6 , MpT'^' p30(f^lt6/+ c-Myb^PM/+ and MpT'^' p30(f"^6/p"⁶ c-Myb^plt4/PU4 or Mpl^+/+ p30(f"^6/+, Mpl^+/+ p30(f"^6/p"⁶, Mpl^+/+ p30(f^!t6/+ c-Myb^PM/+, Mpl^+/+ p30(f"^6/p"⁶ c-Myb^PM/PM, ⁺ and c-Myb^plt4/PM or offspring or back crosses thereof.

21. The animal model of Claim 20 wherein the animal is a rodent.

22. A c-Myb/p300 antagonist for use in the treatment or amelioration of thrombocytopenia

23. A method of identifying a candidate agent that enhances megakaryocytopoiesis and/or haematopoiesis, said method comprising:

i) contacting the candidate agent with a system comprising a c-Myb/p300 complex or a component thereof, or a genetic sequence capable of producing same; and ii) determining the presence of a complex between the agent and the complex or a component thereof or a genetic sequence capable of producing same, a change in activity of the complex or a component thereof or a genetic sequence capable of producing same, or a change in the level of an indicator of the activity of the complex or a component thereof or a genetic sequence capable of producing same.

24. The method of claim 23, wherein the system is a cellular system.