# Patent

## Title
Modulation of beta-catenin coactivator interactions to effect stem cell growth or differentiation

## Abstract

Methods and agents are disclosed for modulating the interaction of β-catenin or γ-catenin with CBP or p300. Agents that increase the binding of CBP to β-catenin are associated with enhancing the β-catenin-related proliferation of adult stem cells, including hematopoietic stem cells, neural stem cells, skin stem cells, and pancreatic stem cells.

## Background
RELATED APPLICATIONS
The present invention claims benefit of priority from U.S. Provisional Patent Application Ser. No. 60/663,363 filed Mar. 18, 2005, under 35 U.S.C. § 119. The foregoing provisional patent application is incorporated herein by reference in its entirety.


BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to compounds and methods for modulating the interaction between β-catenin or γ-catenin and the coactivator protein CBP, or β-catenin or γ-catenin and the coactivator protein p300, to promote proliferation/dedifferentiation or differentiation of stem/progenitor cells.
2. Description of the Related Art
Stem cells have received significant interest over the last few years due to their potential, under suitable cellular microenvironments, to differentiate and develop into a wide array of cell and tissue types. Several important biomedical applications would be enabled by the ability to generate sufficient pools of adult stem cells, including cell replacement therapy, gene therapy, and tissue engineering. According to the National Institutes of Health, the therapeutic use of stem cells will become a cornerstone of medicine within the next two decades:

    
    
        Given the enormous potential of stem cells to the development of new therapies for the most devastating diseases, when a readily available source of stem cells is identified, it is not too unrealistic to say that this research will revolutionize the practice of medicine and improve the quality and length of life (National Institutes of Health. Stem Cells: Scientific Progress and Future Research Directions. Jun. 17, 2001.). However, the development of such applications for adult stem cells has been severely impaired due to the inability to propagate and expand functional adult stem cells in culture. To date, this has proven to be a singular challenge in stem cell research (Sherley, J. (2002) Stem Cells, 20:561-572). For decades, scientists have attempted to grow stem cells in culture to increase the number of cells for transplantation. The challenge of this undertaking lies in the stem cell's predisposition to differentiate. This problem may be associated with the inherent asymmetric cell kinetics of stem cells in postnatal somatic tissues (Sherley, J. (2002) Stem Cells, 20:561-572). Existing scientific methods used for increasing the number of stem cells include culturing cells on 2-D stromal layers and growing them in the presence of various cytokine cocktails (Rebel, V I., et al. (1994) Blood, 83(1):128-136). However, none of the existing ex vivo methods can prevent differentiation of stem cells while promoting proliferation (Rebel, V I. et al. (1996) J Hematother. 5(1):25-37). There is therefore a need in the art for compounds and methods for use in propagating and expanding adult stem cells in culture.
    
    


BRIEF SUMMARY OF THE INVENTION
The invention relates to compounds and methods for modulating the interaction between β-catenin (or γ-catenin) and the coactivator proteins CBP and p300 to either promote proliferation/or differentiation of stem/progenitor cells.
The compounds of the present invention either increase or decrease post-translational modifications (i.e. phosphorylation, acetylation, sulfonylation, glycosylation) of CBP, p300, or β- or γ-catenin thereby modulating the selection of coactivator usage by catenin.
The invention also relates to methods for enhancing the proliferation of mammalian stem cells, by administering to the stem cells an agent that selectively modulates the interaction of β-catenin with CBP or p300.
The invention further relates to a method for modulating the interaction of β-catenin with CBP or p300 in a cell, comprising treating the cell with an agent that affects the post-translational modifications of at least one of CBP or p300 or β-catenin, thereby selectively modulating the interaction of β-catenin with CBP or p300, wherein said agent does not directly bind to CBP or p300.
In an embodiment of this method, the agent increases the binding of β-catenin to CBP.
In a further embodiment of the method, the agent decreases the binding of p300 to β-catenin.
In yet further embodiments of the method, the agent increases the binding of p300 to β-catenin, or the agent decreases the binding of CBP to β-catenin.
The cell may be treated with the agent of the invention ex vivo and the cell may be a stem cell/progenitor cell.
In certain embodiments, the agent is applied topically to a mammal comprising said cell.
In further embodiments the agent increases the binding of β-catenin to the amino-terminal 110 amino acids of CBP or decreases the binding of β-catenin to the amino-terminal 110 amino acids of p300.
In specific embodiments, the agent decreases the binding of β-catenin to the amino-terminal 110 amino acids of p300 by inhibiting the phosphorylation of Ser 89 of p300, such as by a serine protein kinase; the serine protein kinase may be PKC or CaMK. CaMK kinases may phosphorylate different residues in this N-terminal region, for example Ser 89, Ser 24. The protein kinase may also be PAR-1 or PAR-4, acting either directly or via a kinase cascade.
In further specific embodiments, the agent decreases the binding of β-catenin to the amino-terminal 110 amino acids of p300 by inhibiting the phosphorylation of Ser 89 of p300 by increasing the phosphorylation of Ser 90 of p300, such as by a serine protein kinase; the serine protein kinase may be MAPK or CDK.
In other specific embodiments, the agent modulates the interaction of Ser 89 phosphorylated p300 with a 14-3-3 protein, and the agent may be an analog of Fusicoccin, wherein the analog of Fusicoccin has the following general formula:




The invention also relates to a method of modulating the interaction of β-catenin with CBP or p300 in a cell, wherein the agent modulates the interaction of prolyl isomerase (Pin1) with β-catenin, CBP or p300; in certain embodiments, the agent increases the association of Pin1 with CBP.
In all these embodiments, the agent may be incorporated into a biomaterial capable of supporting the growth of a stem cell; the stem cell may be a hematopioetic stem cell.
The invention also relates to a compound having the following general formula (I) and uses thereof to modulate stem cell proliferation, in particular to enhance proliferation:




    
    
        wherein A is —(CHR3)—(C═O)—. B is —(NR4)—, D is —(CHR5)— or —(C═O)—, E is —(ZR6)—, —(C═O)—, G is —(XR7)n—, —(CHR7)—(NR8)—, —(C═O)—(XR9)—, or —(C═O)—, W is —Y(C═O)—, —(C═O)NH—, —(SO2)— or nothing, Y is oxygen or sulfur, X and Z is independently nitrogen or CH, n=o or 1; and R1, R3, R4, R5, R6, R7, R8, R9, R10, and R11 are the same or different and independently selected from an amino acid side chain moiety or derivative thereof, the remainder of the molecule, a linker and a solid support, and stereoisomers thereof. R2 is selected from a monocyclic aryl or heteroaryl moiety bearing the substituent NR10R11, wherein the compound binds preferentially to p300 phosphorylated at Ser89.
    
    


The invention further relates to a method of enhancing the proliferation of a mammalian stem cell, comprising administering to the stem cell an agent that selectively modulates the interaction of β-catenin with CBP or p300; the agent may increase the binding of β-catenin to CBP; and the agent may decrease the binding of β-catenin to p300.
The administration to the stem cell may be ex vivo, and the stem cells may be hematopoietic stem cells, hair cells, neural stem cells, or pancreatic islet cells.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic representation of TGF/β-catenin transcription.
FIG. 2 is a schematic representation of two possible mechanisms of action of β-catenin, resulting from alternative interaction with CBP or p300 in the nucleus.
FIG. 3 illustrates that ICG-001 at 10 μM concentration induces the differentiation of C2C12 myeloblasts, as compared to differentiation medium or growth medium.
FIG. 4 illustrates that differentiation of C2C12 myeloblasts is induced by 10 μM ICG-001 as compared to differentiation medium.
FIG. 5A illustrates the structure of Fusicoccin A (FC-A). FIG. 5B illustrates the structure of Fusicoccin J (FC-J). FIG. 5C illustrates the structure of cotylenin A (CN-A).



DETAILED DESCRIPTION OF THE INVENTION
Stem cells are responsible for the regeneration and maintenance of tissues by balancing the processes of self-renewal (i.e., making new stem cells) and differentiation (i.e., generating cells committed to terminal differentiation). This balance results from integration of regulatory signals intrinsic to the stem cell, as well as extrinsic signals from the microenvironment. Perturbations in the balance between self-renewal and differentiation may result in disease, either as a result of stem cell depletion (e.g., aplastic anemia) or increased self-renewal (e.g., cancer).
Most knowledge about the molecular mechanisms of stem cell regulation in mammals has been derived from studies of the hematopoietic system. There is an extensive and expanding understanding of the molecular mechanisms that regulate differentiation along the terminal lineages. However, a mechanistic understanding of the mechanisms that regulate hematopoietic stem cell (HSC) fate decisions is less well understood. A few genes have been identified that, when deleted, result in perturbation of HSC self-renewal (e.g. TNFα-p55-Receptor, p21, Rae28, and Bmi-1) or altered differentiation (e.g., TEL, PU.1, Flt-3, and p27). HoxB4, β-catenin, and Notch signaling, on the other hand, stimulate HSC self-renewal when over-expressed in HSCs.
Recent work has demonstrated that CBP and p300 play important roles in HSC self-renewal and differentiation. CBP and p300 function as molecular integrators of various transcriptional signals. When recruited to promoters by transcription factors, they function as co-activators of transcription through multiple mechanisms, including chromatin remodeling, acetylation of associated proteins, and recruitment of the basal transcription machinery. CBP and p300 are highly homologous on a structural level, with up to 93% identity within certain protein-binding domains (SEQ ID NO:1 and 2). For most functions, the two proteins appear to be functionally redundant. However, mouse genetic loss-of-function studies demonstrated a difference between p300 and CBP function in HSCs: loss of CBP results in defective HSC self-renewal, whereas loss of p300 results in defective hematopoietic differentiation.
CBP and p300 have been previously shown to interact with many of the known transcription factors shown to be important in HSC regulation (e.g., HoxB4, β-catenin, Notch, AML-1, MLL). Earlier results suggest that within HSCs there may be transcription factors that are specifically co-activated by CBP that are critical for self-renewal, and others that are preferentially co-activated by p300 that are critically required for differentiation. One example of a signaling pathway that seems to utilize CBP and p300 differentially is the Wnt signaling pathway. The Wnt signaling pathway has been shown to play a pertinent role in the development and maintenance of various tissues, including blood, intestines, and skin. Its effects are executed at the level of stem and progenitor cells, affecting both self-renewal and differentiation. Moreover, the importance of Wnt signaling in maintaining the undifferentiated features of embryonic stem (ES) cells has been well established. Importantly, when Wnt signaling is perturbed it can lead to the development of cancer in these same tissues.
β-catenin (SEQ ID NO:3) is a vertebrate homolog of Drosophila gene armadillo, which functions in both cell adhesion and, as discussed herein, the Wnt signaling pathway. γ-catenin (SEQ ID NO:4) is also a vertebrate homolog of armadillo. β-catenin and γ-catenin have analogous structures and functions, and they have the ability to be regulated by the APC tumor suppressor.
Activation of the Wnt signaling pathway requires the nuclear stabilization of TCF (T cell actor)/β-catenin complexes and recruitment of transcriptional co-activators, such as CBP and p300. β-catenin is constitutively produced in the cell, and inhibitory mechanisms exist to maintain β-catenin levels at below those that would lead to aberrant transcriptional activity in vivo, leading to pathological conditions such as cancer. In one example of aberrant regulation, Emami and colleagues (PNAS101: 12682-7, 2004) recently demonstrated that β-catenin preferentially associates with CBP in cancer cells. However, when β-catenin was prevented from associating with CBP, by utilizing a β-catenin/CBP-specific inhibitor, β-catenin could bind to p300. The “alternative” binding of β-catenin to p300 was accompanied by the execution of a differentiative genetic program (Teo J et al. PNAS, 102, 2005). Thus, β-catenin is thought to promote proliferation without differentiation by binding to and activating CBP, and to initiate differentiation with limited proliferation by binding to and activating p300. Perturbation of β-catenin interaction with CBP and/or p300 is expected therefore to influence differentiation or proliferation.
Stem cell therapy is based on the ability of human fetal or adult pluripotent stem cells to differentiate into a variety of cell types. Stem cells may be used to replace damaged cells as a treatment for many different diseases including cancer, Parkinson's disease, spinal cord injury, burns, diabetes, heart disease, rheumatoid arthritis, and osteoarthritis and for gene therapy (Lazic, S E. et al. J Hematother Stem Cell Res, 12(6):635-642, Gafni, Y. et al. Gene Ther. 11(4):417-426). Stem cell therapy has long been an exciting potential medical breakthrough. The ability to inject normal stem cells into a patient, where they could generate organ-specific cells to potentially replace defective patient tissues, offers enormous potential.
The ability to maintain adult skin stem cells in vitro has allowed engraftment of cultured skin onto burn victims (Green, H. (1991) Sci. Am. 265:96-102). Additionally, at present, there are three adult stem cell related transplantation procedures used for hematopoietic reconstititution: bone marrow transplantation (BMT), peripheral blood stem cell transplantation (PBSCT) and umbilical cord blood stem cell transplantation (UCBSCT). The first two hematopoietic reconstitution techniques, BMT and PBSCT, suffer from a significant matching problem with allogeneic donors. The degree of match required for a successful transplant appears to be less stringent for UCBSCT than BMT or PBSCT. However, the relatively lower volume of harvested stem cells and the availability of only one collected cord blood unit per transplant procedure limit the wide applicability of UCBSCT (McCaffrey, P. Lancet Oncol., 6(1): 5, 2005). One solution to this problem is ex vivo expansion of the cord blood stem cells. However, there is a significant hurdle to overcome in order to provide this straightforward solution.
Stem Cells and Cancer “Stem Cells”
A unifying feature of all cancers is their capacity for unlimited self-renewal, which is also a defining characteristic of normal stem cells. Decades ago, it was discovered that the proliferative capacity of all cancer cells was not equivalent, and only a small minority of tumor cells were able to proliferate extensively (Hamburger, A. W. et al. (1977) Science, 197(4302):461-463). This gave rise to the concept that malignant tumors are comprised of Cancer Stem Cells, which have great proliferative potential, as well as another pool of more differentiated cancer cells, with limited proliferative capacity. An important implication of the Cancer Stem Cell hypothesis is that there are mechanistic similarities between the self-renewal of normal stem cells and the proliferation of cancer stem cells (Pardal, R. et al. (2003) Nat. Rev. Cancer 3(12):895-902). Recent studies have demonstrated that specific gene products regulate both the self-renewal of normal somatic stem cells, as well as the proliferation of cancer cells (Park, I. K. et al. (2003) Nature 423:302-305; Lessard, J. et al. (2003) Nature, 423(6937):255-260). This implies that similar mechanisms are utilized in both stem cells and cancer cells to maintain a proliferative, non-differentiated state.
Wnt Signaling in Stem Cells and Cancer
The Wnt/β-catenin pathway initiates a signaling cascade critical in both normal development and the initiation and progression of cancer (Giles, R H et al. (2003) Biochim Biophys Acta, 1653(1):1-24; Wodarz, A. et al. (1998) Annu Rev Cell Dev Biol, 14:59-88). Wnt signaling and in particular the nuclear functions of β-catenin have been shown to be important in the maintenance, proliferation as well as the differentiation of stem cells (Song, X. et al. (2003) Development, 130(14):3259-3268). Some of the salient features of this signaling pathway, relevant to this invention, are summarized in FIG. 1. The Wnt/β-catenin pathway normally regulates expression of a range of genes involved in promoting both proliferation and differentiation. Activation of the Wnt pathway allows β-catenin to accumulate in the nucleus, bind to members of the TCF family of transcription factors, and form a transcriptionally active complex, by recruiting either the transcriptional coactivator CBP or its closely related homolog, p300. However, in greater than 85% of colon cancers, mutations in this pathway lead to constitutive activation and expression of target genes, e.g. c-myc, cyclin D1 and survivin, all of which are critical for rapid cell proliferation (Kolligs, F T. et al. (1999) Mol Cell Biol, 19(8):5696-5706; Tetsu, O. et al. (1999) Nature, 398(6726):422-426; Kim, P J. et al. (2003) Lancet, 362:205-209). Thus, tumorigenesis in the intestinal epithelium appears to be caused by Wnt/β-catenin induced hyper-proliferation of intestinal crypt stem cells, followed by accumulation of additional mutations that confer malignancy and cancer progression. Wnt signaling has also been demonstrated to be important for the maintenance of pluripotency in both mouse and human embryonic stem cells in culture (Sato, N. et al. (2004) Nat Med, 10(1):55-63). Expression of multiple components of the Wnt pathway is evident in the P19 human embryonal carcinoma cell lines, as well as in embryonic stem cells (Walsh, J. et al. (2003) APMIS, 111(1):197-211).
Wnt and Hematopoietic Stem Cells (HSC)
The self-renewal of hematopoietic stem cells (HSC) is also promoted by Wnt signaling. Overexpression of stabilized β-catenin in cultured bone marrow HSC from mice increased the number of these cells in long-term culture as measured by their ability to reconstitute the hematopoietic systems of mice following irradiation. Additionally, purified Wnt3a promoted self-renewal but only partially inhibited the differentiation of HSC in culture (Reya, T. et al. (2003) Nature, 423(6938):409-414).
Differential Coactivator Usage in Wnt/β-catenin Signaling
As discussed above, the functions of CBP and p300 have been described as redundant in several studies (reviewed in Goodman, R H. et al. (2000) Genes Dev, 14(13):1553-1577) and their expression pattern during mouse development is almost identical (Partanen, A. et al. (1999) Int JDev Biol, 43(6):487-494). However, it is becoming increasingly clear that these highly homologous coactivators are not redundant under physiological conditions, and are responsible for distinct transcriptional programs. Rebel et al. (Rebel, V I. et al. (2003) Proc Natl Acad Sci USA, 99(23):14789-14794), using cells from knockout mice, demonstrated that a full dose of CBP, but not p300, is crucial for HSC self-renewal. Conversely, p300 but not CBP, is essential for proper hematopoietic differentiation. Similarly, Eckner and colleagues (Roth, J F. et al. (2003) Embo J, 22(19):5186-5196) demonstrated a critical role for p300's histone acetyltransferase activity (HAT) but not CBP's HAT activities. These studies and others clearly demonstrate that CBP and p300 play non-redundant and distinct roles during development.
From our chemogenomic studies with the small molecule inhibitor of the β-catenin/CBP interaction and additional gene expression profiling, we have developed a model that describes how differential coactivator usage in Wnt signaling controls proliferation vs. differentiation. The critical feature of this model is that the CBP arm (FIG. 2, left side) of the pathway is essential for proliferation without differentiation, for example in cancer or stem cells, whereas the p300 arm (FIG. 2, right side) is critical for differentiation, with limited proliferation. ICG-001 specifically inhibits β-catenin/CBP dependent transcription (i.e. the left arm of the pathway), thus selectively inducing programmed cell death in cancer cells (Emami, K H. et al. (2004) Proc Natl Acad Sci USA, 101, 12682-7, 2004), and inducing the differentiation of non-tumorigenic precursor cells, e.g. C2C12 myoblasts (FIGS. 3, 4) and 3T3-L1 preadipocytes.
Without being bound by a specific mechanism, the invention is based on the premise that selectively inhibiting or down-modulating the β-catenin/p300 interaction (i.e. the right side of the pathway, FIG. 2) allows for proliferation without differentiation of pluripotent stem cells.
For maintenance of hematopoietic stem cell proliferation, a preferable agent of the invention is capable of affecting the post-translational modifications of any one of CBP, p300, or β-catenin, leading to a selective increase of β-catenin interaction with CBP or a selective decrease of β-catenin interaction with p300, wherein the agent does not directly bind to CBP or p300. In one embodiment, the agent increases the binding of β-catenin to CBP. In another embodiment, the agent decreases the binding of β-catenin to p300. With either of these embodiments, the overall result biases the B-catenin pathway towards “proliferative/non-differentitive program” of the target cells, which according to the invention are adult stem cells, such as hematopoietic stem cells, neural stem cells, or skin stem cells. For example, with reference to FIG. 2, preferential binding of β-catenin to CBP, with less binding to p300, is associated with maintaining hematopoietic stem cells in an undifferentiated state wherein they undergo continuous proliferation, resulting in enhanced numbers of undifferentiated cells useful for repopulating the hematopoietic system of a mammal, such as a human, in need of such treatment.
Agents suitable for use according to the invention can be screened using co-immunoprecipitation methods as described in Emami et al. PNAS101:12682-7, 2004. Briefly, target cells, in this case HSC, are transfected with full-length β-catenin or with full-length p300. Nuclear lysates are treated with a radiolabeled test agent alone, or with cold test agent. Unbound radiolabeled test agent is removed, and incorporation of the radiolabeled test agent is measured. The results indicate whether the test agent specifically interacts with p300.
A separate series of experiments can demonstrate inhibition of the interaction of β-catenin with p300. The minimal binding domain of CBP (amino acids 1-111), p300 (amino acids 1-111) and the C-terminal region of β-catenin (SEQ ID NO:3) (amino acids 647-781) are expressed in mammalian cells treated with the appropriate agents to modify the interaction and purified. β-catenin is bound to protein A-agarose beads coated with β-catenin-specific antibody and incubated with either CBP or p300. Unbound proteins are removed by washing, then the specific interactions between β-catenin and p300, and β-cateninand CBP, are challenged using the test agent, for testing the compounds which directly bind to CBP or phosphor Ser89 p300. Agents that either increase the binding of β-catenin to CBP or decrease the binding of β-catenin to p300 are further tested in vitro using a suitable model of hematopoietic stem cell proliferation/differentiation. One such model is described in Rebel, V. I. et al., PNAS 99:14789-14794, 2002.
Agents according to the invention may achieve the desired biological effects through one of several mechanisms. For example, the agent may increase the binding of β-catenin to the amino-terminal 110 amino acids of CBP, or it may decrease the binding of β-catenin to the amino-terminal 110 amino acids of p300. The decrease in binding of β-catenin to p300 may be achieved by inhibiting the phosphorylation of Ser 89 of p300, wherein the phosphorylation is catalyzed by protein kinase C-epsilon (PKC), calcium/calmodulin-dependent protein kinase (CaMK), LKB (PAR-4), AMP activated kinase (PAR-1), or other serine/threonine protein kinase either directly or indirectly via a kinase cascade.
The decreased phosphorylation of Ser 89 of p300 may be achieved by increasing the phosphorylation of Ser 90, for example by mitogen-activated protein kinase 4 (MAPK), cyclin-dependent kinase (CDK), or other serine/threonine protein kinase (eg. PI3K).
A preferable agent of the invention may modulate the interaction of Ser 89-phosphorylated p300 with 14-3-3 proteins. Such agents may be analogs of Fusicoccin, such as Fusicoccin A, Fusicoccin J, and cotylenin A. The structures of Fusicoccin A, Fusicoccin J, and cotylenin A are shown in FIG. 5. Fusicoccin is a fungal toxin that is used to study H+-ATPase activation. The mechanism involves inducing an irreversible bond between the C-terminal portion of H+-ATPase, and 14-3-3 protein. (Svennilid, F. et al., Plant Cell 11:2379-2392, 1999.) As a result, the C-terminal auto-inhibiting domain is displaced. Similarly, analogs of Fusicoccin may modulate the interaction of Ser-89 phosphorylated p300 with 14-3-3 proteins, resulting in the decreased interaction of p300 with β-catenin.
In other embodiments, the agent modulates the interaction of Pin1 with β-catenin or with CBP or p300. In one embodiment of the invention, the agent increases the association of Pin1 with β-Catenin/CBP. Pin1 (prolyl isomerase) has been implicated in cancer mechanisms by inhibiting the interaction of β-catenin with the tumor suppressor APC. Pin1 overexpression has been reported to occur in human breast cancer. (Ryo, A. et al., Nat. Cell Biol. 3:793-801 (2001)). Pin1 has also been implicated in normal spermatogenesis. Atchison, F. W. et al. (Biol. Reprod. 69:1989-1997, 2003) reported that adult Pin1-deficient mice exhibited evidence of accelerated exhaustion of stem cell potential, and possible bias towards the differentiation pathway in the absence of Pin1.
Phosphorylation affects the conformation of proteins and creates conditions for binding of signal transducers to certain suitable domains capable of recognizing the phosphorylated residue or residues. Pin1 specifically recognizes phosphorylated S/T—P bonds (Ser/Thr-Pro motifs). For example, Pin1 directly binds a phosphorylated Ser-Pro motif (Ser 246-Pro) next to the APC-binding site in β-catenin, inhibits β-catenin interaction with adenomatous polyposis coli protein (APC), and thereby increases its translocation into the nucleus. (Ryo, A. et al., Nature Cell Biol. 3:793-801, 2001.)
Pin1 can also affect coactivator interactions with transcription factors. P73 is a transcription factor related to the tumor suppressor p53. Pin1-modified p73 displayed a higher affinity for p300 than unmodified p73. (Montovani, F. et al., Mol. Cell. 14:625-636, 2004.) Similarly, Pin1 binding to phosphorylated β-catenin can increase the β-catenin/CBP interaction and thereby β-catenin/CBP dependent gene transcription promoting proliferation at the expense of differentiation.
The agents of the invention can be incorporated into biomaterials on which hematopoietic stem cells are grown. Examples are disclosed in Horak et al. Biomaterials 25, 5249-60, 2004 and Harrison et al. Biomaterials 25, 4977-86, 2004.
Although hematopietic stem cells are disclosed herein as an embodiment of a target for the methods of the invention, the methods are applicable to any adult mammalian stem cells (or ES cells) that can be used for tissue regeneration. Adult stem cells constitute an undifferentiated population of cells that retain the ability to proliferate throughout postnatal life and to differentiate into specialized cells to replace cells that become diseased, die or are lost. (Agrawal, S. et al; Trends in Biotechnology 23:78-83, 2005.) In addition to HSC, stem cells suitable for use according to the invention include neural stem cells, skin stem cells, muscle stem cells, and pancreatic islet cells.
The goal of diabetes treatment is to restore normal numbers and function of insulin-producing β cells. Trucco, M. (J. Clin. Invest. 115:5-12, 2005) discusses the existence of adult pancreatic precursor cells that can generate β cells, and are referred to as pancreas-derived multipotent precursors. Other stem cells may be induced to direct their differentiation toward the β cell. For either of these sources of β cells, the methods and agents of the invention are suitable for inducing proliferation and limiting differentiation, in order to achieve a suitable number of cells for therapeutic use.
Adult neural stem cells can differentiate into neurons, astrocytes, and oligodendrocytes, which are the three major lineages of the adult nervous system. For such applications of the invention, it may be appropriate to manipulate adult neural stem cells in situ in order to achieve neurogeneration in vivo. Active stem cells exist in adult brain in the dentate gyrus region of the hippocampus and the subventricular zone of the forebrain, and these stem cells can differentiate into neurons, astrocytes and oligodendrocytes. In addition, quiescent stem cell pools exist in the spinal cord, substantia nigra, optic nerve, and hypothalmus. (Agrawal, S. et al., 2005). Thus, defined pools of neural stem cells are available for modulation according to the invention.
Skin stem cells may be induced to proliferate in vivo in order to enhance or restore hair growth. Recent evidence suggests that the Wnt pathway is involved in the ability of skin epithelial cells to acquire and/or maintain characteristics of multipotent stem cells. (Alonso, L. et al.; PNAS 100:11830-11835, 2003). Multipotent stem cells in skin receive Wnt signals before they commit to form hair follicles. In transgenic mouse skin in which β-catenin is constitutively stabilized, adult interfollicular epidermis takes on characteristics of embryonic skin, and may have the capacity to develop into hair follicles. (Gat, V., Cell 95:605-614, 1998). Thus, agents and methods of the invention are suitable for enhancing the proliferation of multipotent stem cells in the skin, to provide a reservoir of cells capable of forming hair follicles in order to increase or replace lost hair growth, including in vivo applications, for example by topical use. U.S. Pat. No. 6,419,913 discloses compositions suitable for topical delivery of therapeutic agents including agents for treatment of hair loss. U.S. Pat. No. 6,680,344 also discloses topical delivery of agents for treating hair loss.
In addition to using stem cells following proliferation induced by the agents and methods of the invention, it is also feasible to alter the stem cells prior to use, by gene therapy. The invention therefore provides methods to enhance the proliferation of mammalian stem cells expressing an exogenous gene, prior to administration of the cells for therapeutic use. The gene therapy may also be conducted in vivo, for example, to alter the differentiation potential of neural stem cells. (Gomes, W. A. et al., Dev. Biol. 255:164-177, 2003; Pardridge, W. M., Curr. Opin. Drug Discov. Devel. 6:683-691, 2003.)
An assay suitable for determining whether mammalian stem cells are maintained in a non-differentiated state involves the use of a reporter gene under the control of the OCT4 promoter. OCT4 is a known marker of the undifferentiated stem/progenitor cell state, and the promoter region can be functionally linked to a reporter gene such as EGFP (enhanced green fluorescent protein) as described in Gerrard, L. et al., Stem Cells 23:124-133 (2005), or luciferase. Using either reporter gene, cells are transfected with an OCT4-reporter gene construct using methods described in Gerrard et al. (2005) and the effect of agents according to the invention on the undifferentiated versus differentiation state of the cells is tested.
Methods for testing the effect of small molecules on stem cells in vitro include those described by Chen, J. K. et al., P.N.A.S. 99:14701-14076 (2002) and Frank-Kamenetsky, M. et al., J. Biol 1:10 (2002).
Agents for use in the invention include a α-helix mimetic structure having the following formula (I):




wherein A is —(C═O)—(CHR3)—, B is —N—R4—, D is —(CHR5)— or —(C═O)—, E is —(ZR6)— or —(C═O)—, G is —(XR7)n—, —(CHR7)—(NR8)—, —(C═O)—(XR9)—, or —(C═O)—, W is —Y(C═O)—, —(C═O)NH—, —(SO2)— or nothing, Y is oxygen or sulfur, X and Z is independently nitrogen or CH, n=0 or 1; and R1, R2, R3, R4, R5, R6, R7, R8 and R9, are the same or different and independently selected from an amino acid side chain moiety or derivative thereof, the remainder of the molecule, a linker and a solid support, and stereoisomers thereof.

More specifically, R1, R2, R3, R4, R5, R6, R7, R8 and R9, are independently selected from the group consisting of aminoC2-5alkyl, guanidineC2-5alkyl, C1-4alkylguanidinoC2-5alkyl, diC1-4alkylguanidino-C2-5alkyl, amidinoC2-5alkyl, C1-4alkylamidino C2-5alkyl, diC1-4alkylamidinoC2-5alkyl, C1-3alkoxy, Phenyl, substituted phenyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C1-4alkylamino, C1-4dialkylamino, halogen, perfluoro C1-4alkyl, C1-4alkyl, C1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), benzyl, substituted benzyl (where the substituents on the benzyl are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C1-4alkylamino, C1-4dialkylamino, halogen, perfluoro C1-4alkyl, C1-3alkyl, nitro, carboxy, cyano, sulfuryl or hydroxyl), naphthyl, substituted naphthyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C1-4alkylamino, C1-4dialkylamino, halogen, perfluoro C1-4alkyl, C1-4alkyl, C1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), bisphenyl methyl, substituted bis-phenyl methyl (where the subsitituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C1-4alkylamino, C1-4dialkylamino, halogen, perfluoro C1-4alkyl, C1-4alkyl, C1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyridyl, subsitituted pyridyl, (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C1-4alkylamino, C1-4dialkylamino, halogen, perfluoro C1-4alkyl, C1-4alkyl, C1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyridylC1-4alkyl, substituted pyridylC1-4alkyl (where the pyridine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C1-4alkylamino, C1-4dialkylamino, halogen, perfluoro C1-4alkyl, C1-4alkyl, C1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyrimidylC1-4alkyl, substituted pyrimidylC1-4alkyl (where the pyrimidine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C1-4alkylamino, C1-4dialkylamino, halogen, perfluoro C1-4alkyl, C1-4alkyl, C1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), triazin-2-yl-C1-4alkyl, substituted triazin-2-yl-C1-4alkyl (where the triazine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C1-4alkylamino, C1-4dialkylamino, halogen, perfluoro C1-4alkyl, C1-4alkyl, C1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), imidazoC1-4alkyl, substituted imidazol C1-4alkyl (where the imidazole substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C1-4alkylamino, C1-4dialkylamino, halogen, perfluoro C1-4alkyl, C1-4alkyl, C1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), imidazolinylCalkyl, N-amidinopiperazinyl-N—C0-4alkyl, hydroxyC2-5alkyl, C1-5alkylaminoC2-5alkyl, hydroxyC2-5alkyl, C1-5alkylaminoC2-5alkyl, C1-5dialkylaminoC2-5alkyl, N-amidinopiperidinylC1-4alkyl and 4-aminocyclohexylC0-2alkyl.
In one embodiment, R1, R2, R6 of E, and R7, R8 and R9 of G are the same or different and represent the remainder of the compound, and R3 or A, R4 of B or R5 of D is selected from an amino acid side chain moiety or derivative thereof. As used herein, the term “remainder of the compound” means any moiety, agent, compound, support, molecule, linker, amino acid, peptide or protein covalently attached to the α-helix mimetic structure at R1, R2, R5, R6, R7, R8 and/or R9 positions. This term also includes amino acid side chain moieties and derivatives thereof.
As used herein, the term “amino acid side chain moiety” represents any amino acid side chain moiety present in naturally occurring proteins including (but not limited to) the naturally occurring amino acid side chain moieties identified in Table 1. Other naturally occurring amino acid side chain moieties for use in this invention include (but are not limited to) the side chain moieties of 3,5-dibromotyrosine, 3,5-diiodotyrosine, hydroxylysine, γ-carboxyglutamate, phosphotyrosine and phosphoserine. In addition, glycosylated amino acid side chains may also be used in the practice of this invention, including (but not limited to) glycosylated threonine, serine and asparagine.







TABLE 1







Amino Acid Side Chain Moieties










Amino Acid Side Chain Moiety
Amino Acid







—H
Glycine



—CH3
Alanine



—CH(CH3)2
Valine



—CH2CH(CH3)2
Leucine



—CH(CH3)CH2CH3
Isoleucine



—(CH2)4NH3+
Lysine



—(CH2)3NHC(NH2)NH2+
Arginine




Histidine



—CH2COO−
Aspartic acid



—CH2CH2COO−
Glutamic acid



—CH2CONH2
Asparagine



—CH2CH2CONH2
Glutamine




Phenylalanine




Tyrosine




Tryptophan



—CH2SH
Cysteine



—CH2CH2SCH3
Methionine



—CH2OH
Serine



—CH(OH)CH3
Threonine




Proline




Hydroxyproline










In addition to naturally occurring amino acid side chain moieties, the amino acid side chain moieties of the present invention also include various derivatives thereof. As used herein, a “derivative” of an amino acid side chain moiety includes modifications and/or variations to naturally occurring amino acid side chain moieties. For example, the amino acid side chain moieties of alanine, valine, leucine, isoleucine and pheylalanine may generally be classified as lower chain alkyl, aryl, or arylalkyl moieties. Derivatives of amino acid side chain moieties include other straight chain or brached, cyclic or noncyclic, substitutes or unsubstituted, saturated or unsaturated lower chain alkyl, aryl or arylalkyl moieties.
As used herein, “lower chain alkyl moieties” contain from 1-12 carbon atoms, “lower chain aryl moieties” contain from 6-12 carbon atoms and “lower chain aralkyl moieties” contain from 7-12 carbon atoms. Thus, in one embodiment, the amino acid side chain derivative is selected from a C1-12 alkyl, a C6-12 aryl and a C7-12 arylalkyl, and in a more preferred embodiment, from a C1-7 alkyl, a C6-10 aryl and a C7-11 arylalkyl.
Amino side chain derivatives of this invention further include substituted derivatives of lower chain alkyl, aryl, and arylalkyl moieties, wherein the substituents is selected from (but are not limited to) one or more of the following chemical moieties: —OH, —OR, —COOH, —COOR, —CONH2, —NH2, —NHR, —NRR, —SH, —SR, —SO2R, —SO2H, —SOR and halogen (including F, Cl, Br and I), wherein each occurrence of R is independently selected from straight chain or branched, cyclic or noncyclic, substituted or unsubstituted, saturated or unsaturated lower chain alkyl, aryl, and aralkyl moieties. Moreover, cyclic lower chain alkyl, aryl and arylalkyl moieties of this invention include naphthalene, as well as heterocyclic compounds such as thiophene, pyrrole, furan, imidazole, oxazole, thiazole, pyrazole, 3-pyrroline, pyrrolidine, pyridine, pyrimidine, purine, quinoline, isoquinoline and carbazole. Amino acid side chain derivatives further include heteroalkyl derivatives of the alkyl portion of the lower chain alkyl and aralkyl moieties, including (but not limited to) alkyl and aralkyl phosphonates and silanes.
Representative R1, R2, R5, R6, R7, R8 and R9 moieties specifically include (but are not limited to) —OH, —OR, —COR, —COOR, —CONH2, —CONR, —CONRR, —NH2, —NHR, —NRR, —SO2R and —COSR, wherein each occurrence of R is as defined above.
In a further embodiment, and in addition to being an amino acid side chain moiety or derivative thereof (or the remainder of the compound in the case of R1, R2, R5, R6, R7, R8 and R9), R1, R2, R5, R6, R7, R8 or R9 may be a linker facilitating the linkage of the compound to another moiety or compound. For example, the compounds for use in this invention may be linked to one or more known compounds, such as biotin, for use in diagnostic or screening assay. Furthermore, R1, R2, R5, R6, R7, R8 or R9 may be a linker joining the compound to a solid support (such as a support used in solid phase peptide synthesis) or alternatively, may be the support itself. In this embodiment, linkage to another moiety or compound, or to a solid support, is preferable at the R1, R2, R7 or R8 position, and more preferably at the R1 or R2 position.
In the embodiment wherein A is —(C═O)—CHR3—, B is —N—R4, D is —(C═O)—, E is —(ZR6)—, G is —(C═O)—(XR9)—, the α-helix mimetic compounds for use in this invention have the following general formula (III):




wherein R1, R2, R4, R6, R9, W and X are as defined above, Z is nitrogen or CH (when Z is CH, then X is nitrogen). In a preferred embodiment, R1, R2, R6, and R9 represent the remainder of the compound, and R4 is selected from an amino acid side chain moiety. In a more specific embodiment wherein A is —O—CHR3—, B is —NR4—, D is —(C═O)—, E is —(ZR6)—, Gi is (XR7)n—, the α-helix mimetic compounds for use in this invention have the following formula (IV):





wherein R1, R2, R4, R6, R7, W, X and n are as defined above, and Z is nitrogen or CH (when Z is nitrogen, then n is zero, and when Z is CH, then X is nitrogen and n is not zero). In a preferred embodiment, R1, R2, R6, and R7 represent the remainder of the compound, and R4 is selected from an amino acid side chain moiety. In this case, R6 or R7 may be selected from an amino acid side chain moiety when Z and X are CH, respectively.

The α-helix mimetic structures for use in the present invention may be prepared by utilizing appropriate starting component molecules (herinafter referred to as “component pieces”). Briefly, in the synthesis of α-helix mimetic structures having formula (II), first and second component pieces are coupled to form a combined first-second intermediate, if necessary, third and/or fourth component pieces are coupled to form a combined third-fourth intermediate (or, if commercially available, a single third intermediate may be used), the combined first-second intermediate and third-fourth intermediate (or third intermediate) are then coupled to provide a first-second-third-fourth intermediate (or first-second-third intermediate) which is cyclized to yield the α-helix mimetic structures of this invention. Alternatively, the α-helix mimetic structures of formula (II) may be prepared by sequential coupling of the individual component pieces either stepwise in solution or by solid phase synthesis as commonly practiced in solid phase peptide synthesis.
Within the definition above, a “first component piece” has the following formula S1




Wherein R2 as defined above, and R is a protective group suitable for use in peptide synthesis. Suitable R groups include alkyl groups and, in a preferred embodiment, R is a methyl group. Such first component pieces may be readily synthesized by reductive amination or substitution reaction by displacement of H2N—R2 from CH(OR)2—CHO or CH(OR)2—CH2-Hal (wherein Hal means a halogen atom).

A “second component piece” has the following formula S2:




Where L1 is carboxyl-activation group such as halogen atom, R3, R4 is as defined above, and P is an amino protective group suitable for use in peptide synthesis. Preferred protective groups include t-butyl dimethylsilyl (TBDMS), t-Butyloxycarbonyl (BOC), Methylosycarbonyl (MOC), 9H-Fluorenylmethyloxycarbonyl (FMOC), and allyloxycarbonyl (Alloc). When L is —C(O)NHR, —NHR may be an carboxyl protective group. N-hydrazino amino acids can be readily prepared according to the procedures of Vidal et al. (Tetrahedron Letters 39:8845-8848, 1998). The conversion of these compounds to the second component pieces of this invention may be readily achieved by activation of the carboxylic acid group of the N-protected hydrazino-amino acid. The conversion of these compounds to the second component pieces may be readily achieved by activation of the carboxylic acid group of the N-protected hydrazino-amino acid. Suitable activated carboxylic acid groups include acid halides where X is a halide such as chloride or bromide, acid anhydrides where X is an acyl group such as acetyl, reactive esters such as an N-hydroxysuccinimide esters and pentafluorophenyl esters, and other activated intermediates such as the active intermediate formed in a coupling reaction using a carbodiimide such as dicyclohexylcarbodiimide (DCC).

A “third component piece” has the following formula S3:




where G, E, and L1 are as defined above. Suitable third component pieces are commercially available from a variety of sources or can be prepared by known methods in organic chemistry.

More specifically, the α-helix mimetic structures for use in this invention of formula (II) are synthesized by reacting a first component piece with a second component piece to yield a combined first-second intermediate, followed by either reacting the combined first-second intermediate with third component pieces sequentially to provide a combined first-second-third-fourth intermediate, and the cyclizing this intermediate to yield the α-helix mimetic structure.
The general synthesis of a α-helix having structure I′ may be synthesized by the following technique. A first component piece 1 is coupled with a second component piece 2 by using coupling reagent such as phosgene to yield, after N-deprotection, a combined first-second intermediate 1-2 as illustrated below:




wherein R1, R2, R4, R7.Fmoc, Moc and X are as defined above, and Pol represents a polymeric support.

All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.
It should be understood that the examples and embodiments described herein are for illustrative purposes only and for that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.

## Claims

1. A method for modulating the interaction of gamma-catenin with p300 in a stem cell or a progenitor cell, comprising treating said cell ex vivo with an agent that modulates a post-translational modifications of p300 or but does not directly bind to p300.

2. The method of claim 1 wherein the agent decreases the binding of p300 to gamma-catenin.

3. The method of claim 1 wherein the agent increases the binding of p300 to gamma-catenin.

4. The method of claim 1 wherein the agent decreases the binding of gamma-catenin to the amino-terminal 110 amino acids of p300.

5. The method of claim 1 wherein the agent inhibits phosphorylation of Ser89 of p300.

6. The method of claim 5 wherein said agent is an analog of Fusicoccin.

7. The method of claim 1 wherein the agent is incorporated into a biomaterial capable of supporting the growth of a stem cell.

8. The method of claim 1, wherein the agent is a compound having the following general formula (I):



wherein A is —(CHR3)—(C═O)—, B is —(NR4)—, D is —(CHR5)— or —(C═O)—, E is —(ZR6)—, —(C═O)—, G is —(XR7)n—, —(CHR7)—(NR8)—, —(C═O)—(XR9)—, or —(C═O)—, W is —Y(C═O)—, —(C═O)NH—, —(SO2)— or nothing, Y is oxygen or sulfur, X and Z is independently nitrogen or CH, n=o or 1, R2 is selected from a monocyclic aryl or heteroaryl moiety bearing the substituent NR10R11, and R1, R3, R4, R5, R6, R7, R8, R9, R10, and R11 are the same or different and independently selected from an amino acid side chain moiety or derivative thereof, a linker and a solid support.