WO2009016384A2 - Gsk-3 inhibitors - Google Patents

Abstract

A peptide inhibitor of glycogen synthase kinase-3 (GSK-3) comprising a peptide having between 6 and 50 amino acid residues and an amino acid sequence selected from PYYVNSGYA (SEQ ID No: 7), EPVNPYYVNSGYALAP (SEQ ID No: 6), and REPVNPYYVNSGYALAPATS (SEQ ID No: 5). A peptide inhibitor of GSK-3 comprising a peptide having between 13 and 50 amino acid residues and an amino acid sequence represented by the formula: EPXAZ1XBZ2XC(A/S)P (SEQ ID Nos: 54-61 ), wherein XA represents 2 or 3 amino acid residues which can, independently, be any amino acid residue, wherein XB represents 4 or preferably 5 amino acid residues which can, independently, be any amino acid residue, wherein Xc represents 1 or 2 amino acid residues which can, independently, be any amino acid residue, wherein Z1 and Z2, independently, represent Y, S or F and wherein (A/S) represents either A or S. The peptide is not phosphorylated at a tyrosine residue indicated by Y.

Description

INHIBITORS

The invention relates to inhibitors of glycogen synthase kinase-3 (GSK-3) and to methods of identifying inhibitors of GSK-3.

Protein kinases, the enzymes that phosphorylate protein substrates, are a large family of proteins which play a central role in the regulation of a wide variety of cellular processes and maintaining control over cellular function (Hanks et al 1988). They are key players in the signalling of extracellular events to the cytoplasm and the nucleus, and are involved in regulating the majority of the events relating to the life and death of cells, including mitosis, differentiation and apoptosis.

GSK-3 is a serine/threonine kinase known for its roles in glycogen metabolism and diabetes, in the Wnt signaling pathway, in the immune system, and in neurological disorders (reviewed by Doble & Woodgett, 2003; Frame & Cohen, 2001 ; Grimes & Jope, 2001 ; and Woodgett, 2001). GSK-3 has been shown to be active in most resting cells and is subject to negative regulation by external stimuli. In response to growth factor stimulation, for example, kinases such as Akt inhibit GSK-3 by phosphorylation on serine 9 (Cross et al 1995; Stambolic & Woodgett, 1994). In some instances, GSK-3 has been shown to be activated by agents that promote phosphorylation on tyrosine 216 (Bhat et al 2000). GSK-3 can also be regulated by binding to the proteins Axin, FRAT (Frequently rearranged in advanced T-cell Iymphomas)/GBP and the Kaposi's sarcoma- associated herpesvirus latency-associated nuclear antigen (Fujimuro et al 2003; lkeda et al 1998; Yost et al 1998). GSK-3 has numerous substrates, including a number of transcription factors such as c-Jun, c-myc, C/EBPs (CCAAT enhancer binding proteins) and NF-ATc (nuclear factor of activated T cells). The effects of phosphorylation by GSK- 3 tend to be inhibitory and include promotion of degradation and enhancement of nuclear export (for references see Frame & Cohen, 2001). Thus, inhibition of GSK-3 often results in increased gene expression. However, there are examples where GSK-3 positively regulates gene expression, such as through CREB phosphorylation (Salas ef al 2003).

Inhibition of GSK-3 has been shown to be beneficial in the treatment of type Il diabetes. GSK-3 inhibits glycogen synthase by direct phosphorylation. Upon insulin activation, GSK-3 is inactivated, thereby allowing the activation of glycogen synthase and possibly other insulin-dependent events. Type Il diabetes, otherwise known as Non-insulin Dependent Diabetes Mellitus (NIDDM), is initially characterised by decreased sensitivity to insulin (insulin resistance) and a compensatory elevation in circulating insulin concentrations, which is characterised by hyperinsulinemia and hyperglycemia. Increased insulin levels are caused by increased secretion from the pancreatic beta cells in an attempt to overcome the insulin resistance. The resulting hyperinsulinemia is associated with a variety of cardiovascular complications. As insulin resistance worsens, the demand on the pancreatic beta cells steadily increases until the pancreas can no longer provide adequate levels of insulin, thereby resulting in elevated levels of glucose in the blood. Thus, diabetes causes impaired glucose transport into skeletal muscle and increased hepatic glucose production, in addition to inadequate insulin response. The disorders and conditions associated with hyperglycemia and hyperlipidemia include cardiovascular disease, renal failure, and blindness.

Although the precise molecular mechanism underlying insulin resistance is unknown, defects in downstream components of the insulin signaling pathway may be the cause. Among the downstream components of insulin signaling is glycogen synthase kinase-3 (GSK-3), a serine/threonine kinase that has been recognised as an important signaling molecule in a variety of cellular processes. High activity of GSK-3 impairs insulin action in intact cells (Eldar-Finkelman et al 1997). This impairment results from the phosphorylation of insulin receptor substrate-1 (IRS-1) serine residues by GSK-3. Likewise, increased GSK-3 activity expressed in cells results in suppression of glycogen synthase activity (Eldar-Finkelman et al 1996). GSK-3 activity is significantly increased in epididymal fat tissue of diabetic mice (Eldar-Finkelman et al 1999). Subsequently, increased GSK-3 activity was detected in skeletal muscle of type 2 diabetes patients. Thus, the inhibition of GSK-3 activity may represent a way to increase insulin activity in vivo.

GSK-3 is also considered to be important in the pathogenesis of Alzheimer's disease. GSK-3 was identified as one of the kinases that phosphorylates tau, a microtubule- associated protein, that is responsible for formation of paired helical filaments (PHF), an early characteristic of Alzheimer's disease. Apparently, abnormal hyperphosphorylation of tau is the cause for destabilization of microtubules and PHF formation. Despite the fact that several protein kinases were shown to promote phosphorylation of tau, only GSK-3 phosphorylation directly affected tau ability to promote microtubule self-assembly (Hanger et al 1992; Mandelkow et al 1992; Mulot et al 1994; Mulot et al 1995). Further evidence came from studies of cells overexpressing GSK-3 and from transgenic mice that specifically expressed GSK-3 in brain. In both cases GSK-3 led to generation of the PHF like epitope tau (Lucas et a/ 2001 ). Another mechanism that links GSK-3 with Alzheimer's disease is its role in cell apoptosis. The fact that insulin is a survival factor of neurons and initiates its anti- apoptotic action through activation of PI3 kinase and PKB (Barber et al 2001), suggested that GSK-3, which is negatively regulated by these signaling components, promotes neuronal apoptosis. Studies have indeed confirmed this view and have shown that GSK- 3 is important in apoptosis. Furthermore, its apoptotic function was shown to be independent of PI3 kinase. Overexpression of GSK-3 in PC12 cells caused apoptosis (Pap et al 1998). Activation of GSK-3 in cerebellar granule neurons mediated migration and cell death (Tong et al 2001). In human neuroblastoma SH-SY5Y cells, overexpression of GSK-3 facilitated staurosporine-induced cell apoptosis. Additional studies also indicated that inhibition of GSK-3 rescued cell death. These studies showed that expression of Fratl, a GSK-3β inhibitor, was sufficient to rescue neurons from death induced by inhibition of PI3 kinase (Crowder et al 2000). A number of small molecules, e.g. SB-216763 and SB-415286 (Glaxo SmithKline Pharmaceuticals) have been developed that specifically inhibit GSK-3. Treatment of primary neurons with these compounds protect from neuronal death induced by reduction in PI3 kinase activity (Cross ed al 2001 ).

GSK-3 has also been implicated in affective disorders, such as bipolar disorder and manic depression, based on the findings that lithium, a primary mood stabiliser frequently used in bipolar disease, is a strong inhibitor of GSK-3 at the therapeutic concentration range used in clinics (Klein et al 1996; Stambolic et al 1996; Phiel et al 2001). A series of studies were undertaken to determine if lithium mimics loss of GSK-3 activity in cellular processes, and lithium was shown to cause activation of glycogen synthesis, stabilization and accumulation of β-catenin (Stamboiic et al 1996), induction of axis duplication in Xenopus embryo (Klein et al 1996), and protection from neuronal death (Bijur ef al 2000). These studies indicated that GSK-3 is a major in vivo target of lithium and thus has important implications in therapeutic treatment of affective disorders. One mechanism by which lithium and other GSK3 inhibitors may act to treat bipolar disorder is to increase the survival of neurons subjected to aberrantly high levels of excitation induced by the neurotransmitter, glutamate (Nonaka et al 1998). Glutamate-induced neuronal excitotoxicity is also believed to be major cause of neurodegeneration associated with acute damage, such as in cerebral ischemia, traumatic brain injury and bacterial infection. Furthermore, it is believed that excessive glutamate signaling is a factor in the chronic neuronal damage seen in diseases such as Alzheimer's, Huntington's, Parkinson's, AIDS associated dementia, amyotrophic lateral sclerosis (AML) and multiple sclerosis (MS) (Thomas, 1995). Consequently GSK-3 inhibitors are believed to be a useful treatment in these and other neurodegenerative disorders.

GSK-3 is involved in additional cellular processes including development (He et a/ 1995), oncogenesis (Rubinfeld et al 1996) and protein synthesis (Welsh et al 1993). Importantly, GSK-3 plays a negative role in these pathways. This suggests that GSK-3 is a cellular inhibitor in signaling pathways. Thus, development of specific drug inhibitors for GSK-3 will have important implications in basic research, as well as therapeutic interventions.

Other examples of GSK-3 mediated diseases or conditions include obesity, neurotraumatic injuries such as acute stroke, immune potentiation, baldness or hair loss, atherosclerotic cardiovascular disease, hypertension, polycystic ovary syndrome, ischemia, brain trauma or injury, immunodeficiency, and cancer including prostate cancer (see, for example, WO 00/38675).

While the inhibition of GSK-3 both by lithium chloride (WO 97/41854) and by purine inhibitors (WO 98/16528) has been reported, these inhibitors are not specific for GSK-3. Similarly, an engineered cAMP response element binding protein (CREB), a known substrate of GSK-3, has been described (Fio! et al 1994), as have two peptide inhibitors of GSK-3 (Fiol et al 1990). However, these substrates only nominally inhibit GSK-3 activity.

WO 02/24941 discloses methods for identifying compounds that inhibit the activity of GSK-3 towards phosphate-dependent substrates to a greater extent than towards non- phosphate-dependent substrates.

Kaidanovich-Beilin & Eldar-Finkelman (2006) review various peptide inhibitors of protein kinases and in Table 1 list three synthetic peptide inhibitors of GSK-3, namely L803-mts, FRATtide and GID.

The peptide KEAP PAP PQS (p)P (SEQ ID No: 1) (L803) is a GSK-3β inhibitor derived from the GSK-3 substrate recognition site of HSF-1 which is N-myristoylated (L803-mts) for intracellular activity and is reported to have anti-diabetic, anti-depressant and anti- Parkinson's effects. L803-mts is available from Calbiochem / Merck (Cat. No. 361545) and has the sequence Myr-N-GKEAPPAPPQS(p)P-NH₂ (SEQ ID No: 2). L803-mts is a cell-permeable myristoylated form of GSK-3β peptide inhibitor with a glycine spacer. It acts as a selective, substrate-specific, competitive inhibitor of GSK-3β (IC₅₀ = 40 μM). It has been shown to mimic insulin action, and activate glycogen synthase activity (~2.5- fold) in HEK293 cells and improve glucose tolerance in diabetic mice (Kaidanovich-Beilin & Eldar-Finkelman, 2006).

The peptide SQPETRTGDDDPHRLLQQLVLSGNLIKEAVRRLHSRRLW (SEQ ID No: 3) (FRATtide; Calbiochem Cat. No. 344265) is a GSK-3 inhibitor derived from the carboxy terminus of FRAT-1 which is reported to block phosphorylation of Axin, β-catenin and tau.

The peptide DIHVDPEKFAAELISRLEGVLRDR (SEQ ID No: 4) (GSK-3 interaction domain, GID) is a GSK-3 inhibitor derived from the GSK-3β interacting domain of Axin which is reported to activate Wnt-dependent transcription and prevents nuclear export. As discussed in WO 2006/018633, the GSK-3-binding domain of Axin (GID) is not technically a GSK-3 inhibitor since it has not been shown to inhibit the catalytic activity of GSK-3 kinase. However, it is believed to sequester GSK-3 preventing its interaction with the androgen receptor, and in this way inhibits the activity of GSK-3.

Nevertheless, there is still a need in the art for additional GSK-3 selective inhibitors.

Here we present evidence that both GSK-3α and GSK-3β phosphorylate Axin on tyrosine. For GSK-3β the phosphorylated residues have been identified as residues Y309 and Y315 (residue numbering from rat Axin) in a sequence that resembles the autophosphorylation site in GSK-3β. We have also shown that an unphosphorylated peptide from Axin called AXPEP (REPVNPYYVNSGYALAPATS; SEQ ID No: 5) containing the Axin tyrosine phosphorylation sites inhibits GSK-3β tyrosine kinase activity in vitro, while a tyrosine-phosphorylated form of this peptide (at the underlined residues equivalent to Y309 and Y315) does not. Therefore, the inventor considers that the peptide EPVNPYYVNSGYALAP (SEQ ID No: 6) containing a minimal Axin site, and the peptide PYYVNSGYA (SEQ ID No: 7) containing a superminimal Axin site, would similarly inhibit GSK-3β kinase activity when unphosphorylated at the equivalent tyrosine residues (underlined). In addition, since GSK-3α also phosphorylates Axin on tyrosine, the inventor considers that the same inhibitor peptides will also inhibit GSK-3α kinase activity.

Moreover since the inventor has shown that both GSK-3α and GSK-3β also phosphorylate Axin2 at tyrosine, the inventor considers that the Axin2 tyrosine phosphorylation site (SEPVNPYHVGSGYVFAPATS; SEQ ID No: 8) will also inhibit GSK- 3 kinase activity.

The Axin tyrosine phosphorylation sites Y309 and Y315 are in a sequence resembling the sequence surrounding Y216, the tyrosine autophosphorylation site in the activation loop of GSK-3β (GEPNVSYICSTYYRAPELI; SEQ ID NO: 9). The inventor's experimental results described below suggest that Axin controls GSK-3 activity by inhibiting autophosphorylation, and that this function of Axin is blocked by tyrosine phosphorylation of Axin. Accordingly, the inventor considers that a peptide comprising a minimal GSK3 autophosphorylation sequence such as EPNVSYICSTYYRAP (SEQ ID

No: 10) from the tyrosine autophosphorylation site of GSK-3β would also inhibit GSK-3 kinase activity.

Based upon these sequences, the inventor has derived a minimal consensus sequence (EPX_n=_2-3YX_n=₄-₅YX_n=i-₂(A/S)P; SEQ ID No: 11-18) that is considered to inhibit GSK-3 kinase activity. Searching for this motif, and for the related consensus

(SEQ ID No: 19-22), we identified related peptide sequences from Atrophin-1 (EPHPSVTPTGYHAP (SEQ ID No: 23) and EPSAPSIPTAYQSSP (SEQ

ID No: 24)); Tau (EPPKSGDRSGYSSP; SEQ ID No: 25); "MYBPC3" (EPPNYKALDFSEAP; SEQ ID NO: 26); "KIAA1462" (EPPVYVPPPSYRSP; SEQ ID No:

27); Sp38 (EPHYYYQFTARYHAAP; SEQ ID No: 28); IL-32 (EPGESFCDKSYGAP; SEQ

ID No: 29); DOX4 (EPAPESCPPHPYPLAP; SEQ ID No: 30) and homeobox B3

(EPHPTYJDLSSHHAP; SEQ ID NO: 31). Each of these peptides is considered to have the ability to inhibit GSK-3 kinase activity, especially when unphosphorylated at the underlined tyrosine residues.

Since only the unphosphorylated peptide AxPEP has the ability to inhibit GSK-3 kinase activity, the inventor considers that replacing either or both of the tyrosine residues equivalent to positions Y309 and Y315 with a non-phosphorylatable residue such as phenylalanine (F) would maintain the ability to inhibit GSK-3 kinase activity. Not being phosphorylatable, such modified peptides would have the added benefit of not losing activity in vivo through unwanted phosphorylation. Accordingly, the inventor considers that each of the following peptides (EPVN)PFYVNSGYA(LAP) (SEQ ID NO: 32), (EPVN)PYYVNSGFA(LAP) (SEQ ID NO: 33), (EPVN)PFYVNSGFA(LAP) (SEQ ID NO: 34), VNSGYA(LAP) (SEQ ID No: 35), VNSGFA(LAP) (SEQ ID No: 36), (EPVN)PYYVNSG (SEQ ID NO: 37) and (EPVN)PFYVNSG (SEQ ID No: 38) would also inhibit GSK-3 kinase activity, especially when unphosphorylated at the underlined tyrosine residues (wherein amino acid residues in parentheses are considered to be optional).

Accordingly, a first aspect of the invention provides a peptide inhibitor of glycogen synthase kinase-3 (GSK-3) comprising a peptide having between 6 and 50 amino acid residues and an amino acid sequence selected from:

PYYVNSGYA (SEQ ID No: 7),

EPVNPYYVNSGYALAP (SEQ ID No: 6), REPVNPYYVNSGYALAPATS (SEQ ID NO: 5),

SEPVNPYHVGSGYVFAPATS (SEQ ID NO: 8),

EPNVSYicsRYYR AP (SEQ ID NO: 39),

GEPNVSYiCSTYYR APELi (SEQ ID No: 9),

EPHP SVTPTGYH AP (SEQ ID No: 23), EPSAPSIPTA YQSSP (SEQ ID NO: 24),

EPPK SGDRSGYS SP (SEQ ID No: 25),

GEPPK SGDRSGYS SPGSP (SEQ ID No: 40),

PFYVNSGYA (SEQ ID No: 41),

EPVNPFYVNSGYA (SEQ ID NO: 42), PFYVNSGYALAP (SEQ ID NO: 43),

EPVNPFYVNSGYALAP (SEQ ID NO: 32),

PYYVNSGFA (SEQ ID No: 44),

EPVNPYYVNSGFA (SEQ ID NO: 45),

PYYVNSGFALAP (SEQ ID No: 46), EPVNPYYVNSGFALAP (SEQ ID NO: 33),

PFYVNSGFA (SEQ ID No: 47),

EPVNPFYVNSGFA (SEQ ID NO: 48),

PFYVNSGFALAP (SEQ ID No: 49),

EPVNPFYVNSGFALAP (SEQ ID NO: 34), VNSGYA (SEQ ID No: 50),

VNSGYALAP (SEQ ID No: 35),

VNSGFA (SEQ IDNo: 51),

VNSGFALAP (SEQ ID No: 36),

PYYVNSG (SEQ ID No: 52), EPVNPYYVNSG (SEQ ID NO: 37), PFYVNSG (SEQ ID No: 53), EPVNPFYVNSG (SEQ ID No: 38), EPPN YKALD FSEAP (SEQ ID No: 26), EPPV YVPPPSYR SP (SEQ ID No: 27), EPHYYYQFTARYHAAP (SEQ ID No: 28), EPGESFCDKS YG AP (SEQ ID No: 29), EPAPESCPPHPYPLAP (SEQ ID No: 30) or EPHPTYTDLS SHHAP (SEQ ID No: 31),

wherein the peptide is not phosphorylated at a tyrosine residue indicated by Y. In this list, spaces have been added solely for the purposes of alignment.

In an embodiment, the tyrosine residue at a position equivalent to Y310 is also unphosphorylated.

It may be preferred that each tyrosine residue in the peptide is unphosphorylated.

It is appreciated that the enzyme GSK-3 (EC 2.7.1.37) has two isoforms, GSK-3α and GSK-3β. Except where the context demands otherwise, by GSK-3 we include both GSK- 3α and GSK-3β.

By GSK-3 we include the meaning of a product of a human GSK-3 gene, including naturally occurring variants thereof. The cDNA sequence corresponding to a human GSK-3β mRNA is found in Genbank Accession No. NM_002093. Human GSK-3β includes the amino acid sequence listed in Genbank Accession Nos. NM_002093 and NP_002084, and naturally occurring variants thereof. The cDNA sequence corresponding to a human GSK-3α mRNA is found in Genbank Accession No. NM_019884. Human GSK-3α includes the amino acid sequence listed in Genbank Accession Nos. NM_019884 and NP_063937, and naturally occurring variants thereof. By GSK-3 we also include a homologous gene product from GSK-3 genes from other species, although this is less preferred.

By an inhibitor of GSK-3 we mean that the peptide inhibits the tyrosine kinase activity of purified GSK-3 as measured by:

(a) inhibition of autophosphorylation of GSK-3. It is appreciated that GSK-3 purified in its mature form (e.g., commercially available from NEB) is already fully autophosphorylated on tyrosine and so its autophosphorylation will not be inhibited by the peptide inhibitors described herein. However, purified GSK3 capable of autophosphorylation in vitro can be derived from bacteria expressing mammalian GSK-3 (see Wang et al, 1994), from Sf9 cells infected with a baculovirus expressing GSK-3 (Dajani et al, 2001), or from a rabbit reticulocyte lysate in vitro transcription/translation system (Lochhead et at, 2006); or

(b) inhibition of tyrosine phosphorylation of purified Axin protein, or a purified fragment of Axin that includes the tyrosine phosporylation sites of Axin and the GSK-3 binding site in Axin (e.g., equivalent to residues 298-506 of rat Axin).

Typically, the inhibitor of GSK-3 inhibits these activities of GSK-3 with an IC₅₀ of about 0.1 mM or lower, and more preferably about 80μM or lower.

It is appreciated that the above tyrosine kinase assays are representative of inhibition of the serine kinase activity of GSK-3 because tyrosine autophosphorylation of GSK3 is required for it to be active.

It is preferred if the inhibitor of GSK-3 is selective for GSK-3. By a "selective" inhibitor of GSK-3 we include the meaning that the inhibitor has an IC₅₀ value for human GSK-3 which is lower than for other human protein kinases. Preferably, the GSK-3 selective inhibitor has an IC₅₀ value at least five or ten times lower than for at least one other human protein kinase, and preferably more than 100 or 500 times lower. More preferably, the GSK-3 selective inhibitor has an ICs₀ value more than 1 ,000 or 5,000 times lower than for at least one other human protein kinase. Also preferably, the selective inhibitor of GSK-3 has a lower IC₅₀ value than for at least 2 or 3 or 4 or 5 or at least 10 other human protein kinases. Methods for determining the selectivity of a GSK- 3 inhibitor are described by Ring et al (2003) with respect to 20 different protein kinases, and the at least one other protein kinase may be any one or more of them.

It is preferred if a selective inhibitor of GSK-3 has an IC₅₀ value at least ten times lower than for human CDC2, one of the most closely related kinases, and preferably at least 100, or 500 times lower. More preferably, the GSK-3 selective inhibitor has an IC₅₀ value more than 1,000 or 5,000 times lower for GSK-3 than for human CDC2.

Yet more preferably, the GSK-3 selective inhibitor has an IC₅O value at least five times lower than for all other human protein kinases, and preferably at least 10, 50, 100 or 500 times lower. This aspect of the invention includes a peptide inhibitor of GSK-3 selected from a peptide consisting of an amino acid sequence selected from:

PYYVNSGYA (SEQ ID No: 7),

EPVNPYYVNSGYALAP (SEQ ID No: 6),

REPVNPYYVNSGYALAPATS (SEQ ID NO: 5),

SEPVNPYHVGSGYVFAPATS (SEQ ID NO: 8),

EPNVSYicsRYYR AP (SEQ ID NO: 39), GEPNVSYicsTYYR APELi (SEQ ID No: 9),

EPHP SVTPTGYH AP (SEQ ID No: 23),

EPSAPSiPTA YQSSP (SEQ ID NO: 24),

EPPK SGDRSGYS SP (SEQ ID No: 25),

GEPPK SGDRSGYS SPGSP (SEQ ID No: 40), PFYVNSGYA (SEQ ID No: 41),

EPVNPFYVNSGYA (SEQ ID NO: 42),

PFYVNSGYALAP (SEQ ID NO: 43),

EPVNPFYVNSGYALAP (SEQ ID NO: 32),

PYYVNSGFA (SEQ ID No: 44), EPVNPYYVNSGFA (SEQ ID NO: 45),

PYYVNSGFALAP (SEQ ID NO: 46),

EPVNPYYVNSGFALAP (SEQ ID No: 33),

PFYVNSGFA (SEQ ID No: 47),

EPVNPFYVNSGFA (SEQ ID NO: 48), PFYVNSGFALAP (SEQ ID NO: 49),

EPVNPFYVNSGFALAP (SEQ ID NO: 34),

VNSGYA (SEQ ID No: 50),

VNSGYALAP (SEQ ID No: 35),

VNSGFA (SEQ ID No: 51), VNSGFALAP (SEQ ID No: 36),

PYYVNSG (SEQ ID No: 52),

EPVNPYYVNSG (SEQ ID No: 37),

PFYVNSG (SEQ ID No: 53),

EPVNPFYVNSG (SEQ ID NO: 38), EPPN YKALD FSEAP (SEQ ID No: 26), EPPV YVPPPSYR SP (SEQ ID No: 27), EPHYYYQFTARYHAAP (SEQ ID No: 28), EPGESFCDKS YG AP (SEQ ID No: 29), EPAPESCPPHPYPLAP (SEQ ID No: 30) or EPHPTYTDLS SHHAP (SEQ ID No: 31 ),

wherein the peptide is not phosphorylated at a tyrosine residue indicated by Y. In this list, spaces have been added solely for the purposes of alignment.

In an embodiment, the tyrosine residue at a position equivalent to Y310 is also unphosphorylated. Each tyrosine residue in the peptide may be unphosphorylated.

The invention includes a peptide inhibitor of GSK-3 comprising a peptide having between 13 and 50 amino acid residues and an amino acid sequence represented by the formula: EPXAZ₁XBZ₂XC(AZS)P (SEQ ID Nos: 54-61 ), wherein X_A represents 2 or 3 amino acid residues which can, independently, be any amino acid residue, wherein X₆ represents 4 or 5 amino acid residues which can, independently, be any amino acid residue, wherein Xc represents 1 or 2 amino acid residues which can, independently, be any amino acid residue, wherein Z₁ and Z₂, independently, represent Y, S or F, wherein (A/S) represents either A or S, and wherein the peptide is not phosphorylated at a tyrosine residue indicated by Y.

It is preferred if the peptide inhibitor of GSK-3 is selective for GSK-3 as defined above.

In an embodiment, each independently, Z₁ is preferably Y₁ and Z₂ is preferably Y. Thus, either Z-i , or Z₂, or both of Z-i and Z₂, may be Y.

In an embodiment, each independently, Z-i may be F, and Z₂ may be F. Thus, either Z₁, or Z₂, or both of Z₁ and Z₂, may be F.

In an embodiment, X_A consists of any 2 or all 3 residues selected from residues XiX₂X₃ wherein, each independently, Xi is preferably V₁ X₂ is preferably N and X₃ is preferably P.

In an embodiment, X_B consists of any 4 or all 5 residues selected from residues X₄X₅X₆XyXs (SEQ ID No: 62) wherein, each independently, X₄ is preferably Y, X₅ is preferably V, X₆ is preferably N, X₇ is preferably S and

X₈ is preferably G.

It is, however, preferred that XB represents 5 amino acid residues which can, independently, be any amino acid residue. As a further preference, XB consists of residues X₄X₅X₆X₇X₈ (SEQ ID No: 62) wherein, each independently, X₄ is preferably Y, X₅ is preferably V, X₆ is preferably N, X₇ is preferably S and X₈ is preferably G.

In an embodiment, Xc consists of either one or both residues selected from X₉Xi₀ wherein, each independently, X₉ is preferably A and X₁₀ is preferably L.

In more specific embodiments of this aspect of the invention, the peptide inhibitors of GSK-3 comprise an amino acid sequence selected from:

X₁₁ EPXAZ₁XBZ₂XC(AZS)P (SEQ ID NOS: 63-70), EPXAZIX_BZ₂XC(A/S)PX₁₂ (SEQ ID Nos: 71 -78),

X₁₁ EPXAZ₁XBZ₂XC(AZS)PX₁₂ (SEQ ID NOS: 79-86),

EPXAZ₁XBZ₂XC(AZS)PX₁₂X₁₃ (SEQ ID NOS: 87-94),

X₁₁EPXAZ₁XBZ₂XC(AZS)PX₁₂X₁₃ (SEQ ID NOS: 95-102), EPXAZ₁XBZ₂XC(AZS)PX₁₂X₁₃X₁₄ (SEQ ID NOS: 103-1 10) or X₁₁ EPXAZ₁XBZ₂XC(AZS)PX₁₂X₁₃X₁₄ (SEQ ID NOS: 1 1 1 -1 18), wherein X_1I1 X₁₂, Xi₃ and Xi₄, each independently, represent any amino acid residue. In an embodiment, each independently, Xii is preferably V, X-₁₂ is preferably A₁ X₁₃ is preferably T and X₁₄ is preferably S.

A further specific embodiment of this aspect of the invention provides a peptide inhibitor of GSK-3 comprising a peptide having between 16 and 50 amino acid residues and an amino acid sequence represented by the formula: X₁₁EPX₁X₂X₃Z₁X₄X₅X₆X₇X₈Z₂X₉X₁O(AZS)PX₁₂X₁₃X₁₄ (SEQ ID NO: 119), wherein X-i to X₁₄, each independently, represent any amino acid residue, wherein Z-i and Z₂, each independently, represent Y or F, and wherein the peptide is not phosphorylated at a tyrosine residue indicated by Y.

In this embodiment, each independently,

X_I is preferably V, X₂ is preferably N, X₃ is preferably P, X₄ is preferably Y,

X₅ is preferably V,

X₆ is preferably N,

X₇ is preferably S,

X₈ is preferably G, X₉ is preferably A,

X₁₀ is preferably L,

X_II is preferably V, X₁₂ is preferably A, X-_I3 is preferably T and X₁₄ is preferably S.

Typically, the peptide inhibitor of GSK-3 is less than 30 amino acid residues in length. Thus, the peptide inhibitor of GSK-3 may have a length of from 6 to 10 amino acid residues, or has a length of from 10 to 15 amino acid residues, or has a length of from 15 to 20 amino acid residues, or has a length of from 20 to 25 amino acid residues, or has a length of from 25 to 30 amino acid residues. Peptide inhibitors of about 6 to 10 amino acid residues or greater are sufficient to inhibit GSK-3 activity. Inhibitors of 7-20 amino acid residues in length are preferred, with a length of 10-20 amino acids being more preferred, with 10-13 amino acids being one preferred embodiment.

Alternatively, the peptide inhibitor of GSK-3 may have a length of from 20 to 50 amino acid residues, for example a length of from 20 to 30 amino acid residues, or a length of from 30 to 40 amino acid residues, or a length of from 40 to 50 amino acid residues. As discussed below, these longer peptides typically also comprise additional amino acids in a sequence that facilitates entry into cells (i.e. a cell-penetrating peptide).

Thus, the peptide inhibitor may further comprise the sequence of a cell-penetrating peptide (also known as a protein transduction domain) that facilitates entry into cells. As is well known in the art, cell-penetrating peptides are generally short peptides of up to 30 residues having a net positive charge and act in a receptor-independent and energy- independent manner (Lindgren et al, 2000; Deshayes et al, 2005a and 2005b; Takeuchi et al, 2006, the entire disclosure of which relating to cell-penetrating peptides is incorporated herein by reference).

Among the best characterised cell-penetrating peptides is the Antennapedia-derived peptide (e.g., see Derosssi et al, 1994, 1996; and Prochiantz 1996), which is a 16- residue polypeptide (RQIKIWFQNRRMKWKK; SEQ ID No: 120) corresponding to residues 43-58 (i.e. the third helix of the homeodomain) of Antennapedia, a Drosophila transcription factor. The entire disclosure of Derosssi et al and Prochiantz relating to cell-penetrating peptides is incorporated herein by reference.

An Antennapedia-derived peptide is commercially available as Penetratin™ (http://www.qbiogene.com/products/transfection/penetratin. shtml) from Quantum Biotechnologies. Penetratin™ is a 16-amino acid peptide corresponding to the third helix of the homeodomain of Drosophila Antennapedia (pAntp) protein. This peptide is able to translocate across biological membranes by an energy-independent mechanism. With the use of the Penetratin™ peptide, covalently attached peptides are internalised and conveyed to the cytoplasm and nucleus in a wide variety of cell types.

Console et al (2002) described protein transcription domains derived from Antennapedia (SGRQIKIWFQNRRMKWKKC; SEQ ID No: 121) and HIV-1 TAT (SGYGRKKRRQRRRC; SEQ ID NO: 122) that mediate the uptake of molecules such as polypeptides into cells. The entire disclosure of Console et al relating to cell-penetrating peptides is incorporated herein by reference.

Suzuki et al (2002) described arginine-rich proteins including HIV-1 Rev (34-50) and octoarginine that are efficiently translocated through the cell membrane and act as protein carriers. The sequence of these cell-penetrating peptides is listed in Table 1 of Suzuki et al (2005). The entire disclosure of Suzuki et al relating to cell-penetrating peptides is incorporated herein by reference.

Jones et al (2005) characterised the peptide-mediated delivery of four cell-penetrating peptides, including peptides derived from Antennapedia, TAT, Transportan and a polyarginine peptide. The sequence of these cell-penetrating peptides is listed in Table 1 of Jones et al (2005). The entire disclosure of Jones et al relating to cell-penetrating peptides is incorporated herein by reference.

Further cell-penetrating peptides include the S4i₃-PV and Pep-1 peptides derived from dermaseptin S4 and the SV40 large T nuclear localisation sequence (Mano et al, 2005). The sequence of these cell-penetrating peptides is listed in Table 1 of Mano et al (2005). The entire disclosure of which Mano et al relating to cell-penetrating peptides is incorporated herein by reference.

De Coupade et al (2005) describe ten cell-penetrating peptides of 14-22 residues in length that are able to transport other peptides to the cytoplasm or nucleus of target cells. These peptides, referred to as Vectocell^® penetrating peptides, were derived from superoxide dismutase, platelet-derived growth factor, epidermal-like growth factor, intestinal mucin, CAP37, superoxide dismutase and intestinal mucin, intestinal mucin and PDGF, and apolipoprotein B and anti-DNA antibody. The sequence of the Vectocell^® penetrating peptides is listed in Table 1 of de Coupade et al (2005). The entire disclosure of de Coupade et al relating to cell-penetrating peptides is incorporated herein by reference.

It is appreciated that the sequence of the cell-penetrating peptide may be adjacent to the portion of the peptide that inhibits GSK-3 activity as described above. Alternatively, these two sequences may be separated by one or more amino acids residues, such as glycine residues, acting as a spacer. Typically, the peptide inhibitor of GSK-3 is prepared containing both the sequence of the cell-penetrating peptide and of the peptide that inhibits GSK-3 activity using the methods described below. Alternatively, peptides comprising these two sequences may be combined, for example, by coupling an N-terminal pyridyl disulfide function on one peptide with an available thiol group (i.e. cysteine) on the other peptide. Many other methods for coupling two peptides are known in the art.

In another embodiment, the peptide may be modified for intracellular activity. For example, as described by Kaidanovich-Beilin & Eldar-Finkelman (2006) the peptide may be N-terminally myristoylated, optionally via a glycine spacer. Alternatively, the peptide may be N-terminally stearated.

The peptide may also be modified so that it can be more easily detected, for example by biotinylating it or by linking it to fluorescently-labelled gold nanoparticles.

The peptides are typically made using protein chemistry techniques for example using partial proteolysis (either exolytically or endolytically), or by de novo synthesis. Alternatively, the variants may be made by recombinant DNA technology. Suitable techniques for cloning, manipulation, modification and expression of nucleic acids, and purification of expressed proteins, are well known in the art and are described for example in Sambrook et a/ (2001) "Molecular Cloning, a Laboratory Manual", 3^rd edition, Sambrook et a/ (eds), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.

Preferably, each of the amino acid residues represented by X comprises a naturally occurring amino acid residue which is encoded by DNA, selected from alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (GIn, Q), glutamate (GIu, E), glycine (GIy, G), histidine (His, H), isoleucine (lie, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (VaI, V). However, other when the peptide is made by expression from a polynucleotide, the amino acid residues represented by X may comprise one or more amino acid residues which are not encoded by DNA, including those described below.

The amino acid residues described herein are preferably in the "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the peptide inhibitors retain specificity for GSK-3. This definition includes, unless otherwise specifically indicated, chemically-modified amino acids, including amino acid analogues (such as penicillamine, 3-mercapto-D-valine), naturally- occurring non-proteogenic amino acids (such as norleucine), and chemically-synthesised compounds that have properties known in the art to be characteristic of an amino acid. The term "proteogenic" indicates that the amino acid can be incorporated into a protein in a cell through well-known metabolic pathways.

In other words, the peptide can be a peptide "mimetic". Thus the present invention provides for peptidomimetics which mimic the structural features of the peptide inhibitor of GSK-3 comprising or consisting of an amino acid sequence as described above.

Although most inhibitors of GSK-3 are expected to be peptides, by the use of the screening method and the computer-assisted method of structure-based drug design described below and known in the art, other non-peptide inhibitors of GSK-3 can be identified. The peptidomimetics that are non-peptide in nature can be designed and synthesised by standard organic chemical methods. The peptidomimetics that are non- peptide in nature can be even more advantageous in therapeutic use, in the resistance to degradation, in permeability and in possible oral administration.

Peptidomimetics are small molecules that can bind to proteins by mimicking certain structural aspects of peptides and proteins. They are used extensively in science and medicine as agonists and antagonists of protein and peptide ligands of cellular and other receptors, and as substrates and substrate analogues for enzymes. Some examples are morphine alkaloids (naturally-occurring endorphin analogues), penicillins (semi- synthetic), and HIV protease inhibitors (synthetic). Such compounds have structural features that mimic a peptide or a protein and as such are recognised and bound by other proteins. Binding the peptidomimetic either induces the binding protein to carry out the normal function caused by such binding (agonist) or disrupts such function (antagonist, inhibitor).

A primary goal in the design of peptide mimetics has been to reduce the susceptibility of mimetics to cleavage and inactivation by peptidases. In one approach, such as disclosed by Sherman et al (1990), one or more amide bonds have been replaced in an essentially isosteric manner by a variety of chemical functional groups. This stepwise approach has met with some success in that active analogues have been obtained. In some instances, these analogues have been shown to possess longer biological half- lives than their naturally-occurring counterparts. In another approach, a variety of uncoded or modified amino acids such as D-amino acids and N-methyl amino acids have been used to modify mammalian peptides. Alternatively, a presumed bioactive conformation has been stabilised by a covalent modification, such as cyclization or by incorporation of γ-lactam or other types of bridges (Veber et al, 1978) and Thorsett et al, 1983). Another approach, disclosed by Rich (1986) has been to design peptide mimics through the application of the transition state analogue concept in enzyme inhibitor design. For example, it is known that the secondary alcohol of statine mimics the tetrahedral transition state of the sessile amide bond of the pepsin substrate.

In US 5,552,534, non-peptide compounds are disclosed which mimic or inhibit the chemical and/or biological activity of a variety of peptides. Such compounds can be produced by appending to certain core species, such as the tetrahydropyranyl ring, chemical functional groups which cause the compounds to be at least partially cross- reactive with the peptide. Other techniques for preparing peptidomimetics are disclosed in US 5,550,251 and US 5,288,707.

Commercially available software packages can be used to design small peptides and/or peptidomimetics containing, phosphoserine or phosphothreonine analogues, preferably non-hydrolysable analogues, as specific antagonists/inhibitors. Suitable commercially available software for analyzing crystal structure, designing and optimizing small peptides and peptidomimetics include, but are not limited to: Macromolecular X-ray Crystallography QUANTA Environment (Molecular Simulations, Inc.); TeXsan, BioteX, and SQUASH (Molecular Structure Corporation); and Crystallographica (Oxford Cryostsystems).

The peptide inhibitors of the present invention also include salts and chemical derivatives of the peptides. "Chemical derivative" refers to a polypeptide of the invention having one or more residues chemically derivatised by reaction of a functional side group. Such derivatised molecules include, for example, those molecules in which free amino groups have been derivatised to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatised to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatised to form O-acyl or O-alkyl derivatives. Also included as chemical derivatives are those peptides that contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example, 4-hydroxyproline may be substituted for proline; 5- hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine. The chemical derivatisation does not include changes in functional groups which change one amino acid to another.

Some useful modifications are designed to increase the stability and, therefore, the half- life of the peptide inhibitor in solutions, particularly biological fluids, such as blood, plasma or serum, by blocking proteolytic activity in the blood. A peptide inhibitor can have a stabilising group at one or both termini. Typical stabilising groups include amido, acetyl, benzyl, phenyl, tosyl, alkoxycarbonyl, alkyl carbonyl, benzyloxycarbonyl and the like end group modifications. Additional modifications include using a "L" amino acid in place of a "D" amino acid at the termini, cyclization of the peptide inhibitor, and amide rather than amino or carboxy termini to inhibit exopeptidase activity.

A peptide inhibitor of the invention may or may not be glycosylated. The peptide inhibitors are not glycosylated, for example, when produced directly by peptide synthesis techniques or are produced in a prokaryotic cell transformed with a recombinant polynucleotide. Eukaryotically-produced peptide molecules are typically glycosylated.

The peptide inhibitors of the invention can be produced by well known chemical procedures, such as solution or solid-phase peptide synthesis, or semi-synthesis in solution beginning with protein fragments coupled through conventional solution methods, as described by Dugas et al (1981). Alternatively, a peptide inhibitor of the invention can be synthesised by using well known methods, including recombinant methods and chemical synthesis.

A peptide inhibitor of the invention can be chemically synthesised, for example, by the solid phase peptide synthesis of Merrifield et al (1964). Alternatively, a peptide inhibitor of the invention can be synthesised using standard solution methods (see, for example, Bodanszky, 1984). Newly synthesised peptides can be purified, for example, by high performance liquid chromatography (HPLC), and can be characterised using, for example, mass spectrometry or amino acid sequence analysis.

The peptide inhibitors of the invention may be particularly useful when they are maintained in a constrained secondary conformation. The terms "constrained secondary structure," "stabilised" and "conformationally stabilised" indicate that the peptide bonds comprising the peptide are not able to rotate freely but instead are maintained in a relatively fixed structure. A method for constraining the secondary structure of a newly synthesised linear peptide is to cyclise the peptide using any of various methods well known in the art. For example, a cyclised peptide inhibitor of the invention can be prepared by forming a peptide bond between non-adjacent amino acid residues as described, for example, by Schiller et al (1985). Peptides can be synthesised on the Merrifield resin by assembling the linear peptide chain using N^α-Fmoc-amino acids and Boc and tertiary-butyl proteins. Following the release of the peptide from the resin, a peptide bond can be formed between the amino and carboxy termini. A newly synthesised linear peptide can also be cyclised by the formation of a bond between reactive amino acid side chains. For example, a peptide containing a cysteine-pair can be synthesised, with a disulfide bridge, can be formed by oxidizing a dilute aqueous solution of the peptide with K₃Fe(CN)₆. Alternatively, a lactam such as an ε-(γ-glutamyl)- lysine bond can be formed between lysine and glutamic acid residues, a lysinonorleucine bond can be formed between lysine and leucine residues or a dityrosine bond can be formed between two tyrosine residues. Cyclic peptides can be constructed to contain, for example, four lysine residues, which can form the heterocyclic structure of desmosine (see, for example, Devlin, 1997). Methods for forming these and other bonds are well known in the art and are based on well-known rules of chemical reactivity (Morrison et al, 1992).

A second aspect of the invention provides a polynucleotide encoding a peptide inhibitor of GSK-3 as defined above with respect to the first aspect of the invention.

As is appreciated, the polynucleotides of this aspect of the invention can only encode a peptide consisting entirely of amino acid residues naturally encoded by DNA, and not any of the "non-encoded" amino acids.

The invention also includes a vector comprising a polynucleotide that a peptide inhibitor of GSK-3 as defined in the first aspect of the invention. The vector is typically a plasmid vector or a viral vector. The invention further includes a cell containing the polynucleotide or the vector. The cell is typically a bacterial a yeast cell, or a cell from an insect or mammalian cell line. A large number of suitable vectors and cell lines for cloning and expressing the peptide inhibitor of GSK-3 are very well known in the art. Similarly, a large number of gene therapy vectors suitable for administration to a patient for targeted expression of the peptide inhibitor of GSK-3 are well known in the art.

The polynucleotides, vectors and cells of this aspect of the invention may be useful in gene therapy type applications where inhibition of GSK-3 is therapeutically warranted, in research, and in a method of making the peptide inhibitor of GSK-3 as defined in the first aspect of the invention using recombinant DNA technology. Typically, the method comprises providing a plurality of cells that contain a polynucleotide that encodes the peptide inhibitor of GSK-3 under conditions suitable for expression of the peptide from the polynucleotide encoding it, and obtaining the enzyme thus produced. Typically, the method further comprises isolating and/or purifying the peptide thus obtained. Suitable techniques for cloning, manipulation, modification and expression of nucleic acids, and production, isolation and purification of expressed proteins, are well known in the art and are described for example in Sambrook et al (2001).

A third aspect of the invention provides pharmaceutical composition comprising a peptide inhibitor of GSK-3 according to the first aspect of the invention or a polynucleotide of the second aspect of the invention and a pharmaceutically acceptable excipient, solvent, diluent or carrier (including combinations thereof). The carrier, diluent, solvent or excipient must be "acceptable" in the sense of being compatible with the compounds of the invention (the peptide inhibitors and polynucleotides encoding them) and not deleterious to the recipients thereof. Typically, the carriers will be water or saline which will be sterile and pyrogen free. Suitable excipients include mannitol and dextrose. Acceptable carriers, solvents, diluents and excipients for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro ed. 1985). The choice of pharmaceutical carrier, solvent, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient, solvent or diluent any suitable binder, lubricant, suspending agent, coating agent, or solubilising agent. Preservatives, stabilisers, dyes and even flavouring agents may be provided in the pharmaceutical composition.

The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the peptide inhibitor with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. Formulations in accordance with the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g. povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g. sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide desired release profile.

Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

In a preferred embodiment, the peptide inhibitors of GSK-3 are administered orally. Preferred unit dosage formulations are those containing a daily dose or unit, daily sub- dose or an appropriate fraction thereof, of the GSK-3 inhibitor. For example, with respect to prostate cancer, the Kj for SB216763 is 3 μM and this was found to be the optimal dose for inhibition of prostate cancer growth in CWR-R1 cells. Lower doses of CHIR 98014 were administered to rats (Ring et a/, 2003). The dose of the GSK-3 inhibitor to be administered is one that provides an effective concentration at the prostate cancer of between 0.1 and 10 μM, preferably between 1 and 10 μM.

In addition to the ingredients particularly mentioned above, the formulations may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.

It is appreciated that the GSK-3 inhibitor may be encoded by a polynucleotide. Polynucleotides may be administered by any effective method, for example, parenterally (e.g. intravenously, subcutaneously, intramuscularly) or by oral, nasal or other means which permit the oligonucleotides to access and circulate in the patient's bloodstream. Polynucleotides administered systemicaliy preferably are given in addition to locally administered polynucleotides, but also have utility in the absence of local administration. A dosage in the range of from about 0.1 to about 10 grams per administration to an adult human generally will be effective for this purpose.

The polynucleotide may be administered as a suitable genetic construct as is described below and delivered to the patient where it is expressed. Typically, the polynucleotide in the genetic construct is operatively linked to a promoter which can express the compound in the cell. The genetic constructs of the invention can be prepared using methods well known in the art, for example in Sambrook et al (2001).

Dendritic cell vaccine approaches may be useful in gene therapy for combating prostate cancer.

Although genetic constructs for delivery of polynucleotides can be DNA or RNA it is preferred if it is DNA.

Preferably, the genetic construct is adapted for delivery to a human cell. Means and methods of introducing a genetic construct into a cell in an animal body are known in the art. For example, the constructs of the invention may be introduced into cells by any convenient method, for example methods involving retroviruses, so that the construct is inserted into the genome of the cell. For example, purified retroviruses may be administered (Kuriyama et al (1991) Cell Struc. and Func. 16, 503-510). Retroviral DNA constructs comprising a polynucleotide as described above may be made using methods well known in the art. To produce active retrovirus from such a construct it is usual to use an ecotropic psi2 packaging cell line grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% foetal calf serum (FCS). Transfection of the cell line is conveniently by calcium phosphate co-precipitation, and stable transformants are selected by addition of G418 to a final concentration of 1 mg/ml (assuming the retroviral construct contains a neo^R gene). Independent colonies are isolated and expanded and the culture supernatant removed, filtered through a 0.45 μm pore-size filter and stored at -70°C. For the introduction of the retrovirus into the tumour cells, it is convenient to inject directly retroviral supernatant to which 10 μg/ml Polybrene has been added. For tumours exceeding 10 mm in diameter it is appropriate to inject between 0.1 ml and 1 ml of retroviral supernatant; preferably 0.5 ml. Alternatively, cells which produce retroviruses may be injected. The retrovirus-producing cells so introduced are engineered to actively produce retroviral vector particles so that continuous productions of the vector occurred within the tumour mass in situ. Thus, proliferating epidermal cells can be successfully transduced in vivo if mixed with retroviral vector-producing cells.

Other methods involve simple delivery of the construct into the cell for expression therein either for a limited time or, following integration into the genome, for a longer time. An example of the latter approach includes liposomes (Nassander et al (1992) Cancer Res. 52, 646-653).

Other methods of delivery include adenoviruses carrying external DNA via an antibody- polylysine bridge (see Curiel (1993) Prog. Med. Virol. 40, 1-18) and transferrin-polycation conjugates as carriers (Wagner et al (1990) Proc. Natl. Acad. Sci. USA 87, 3410-3414). In the first of these methods a polycation-antibody complex is formed with the DNA construct or other genetic construct of the invention, wherein the antibody is specific for either wild-type adenovirus or a variant adenovirus in which a new epitope has been introduced which binds the antibody. The polycation moiety binds the DNA via electrostatic interactions with the phosphate backbone. The adenovirus, because it contains unaltered fibre and penton proteins, is internalised into the cell and carries into the cell with it the DNA construct of the invention. It is preferred if the polycation is polylysine. The polynucleotide may also be delivered by adenovirus wherein it is present within the adenovirus particle, for example, as described below.

In an alternative method, a high-efficiency nucleic acid delivery system that uses receptor-mediated endocytosis to carry DNA macromolecules into cells is employed. This is accomplished by conjugating the iron-transport protein transferrin to polycations that bind nucleic acids. Human transferrin, or the chicken homologue conalbumin, or combinations thereof is covalently linked to the small DNA-binding protein protamine or to polylysines of various sizes through a disulfide linkage. These modified transferrin molecules maintain their ability to bind their cognate receptor and to mediate efficient iron transport into the cell. The transferrin-polycation molecules form electrophoretically stable complexes with DNA constructs or other genetic constructs of the invention independent of nucleic acid size (from short oligonucleotides to DNA of 21 kilobase pairs). When complexes of transferrin-polycation and the DNA constructs or other genetic constructs of the invention are supplied to the tumour cells, a high level of expression from the construct in the cells is expected.

High-efficiency receptor-mediated delivery of the DNA constructs or other genetic constructs of the invention using the endosome-disruption activity of defective or chemically inactivated adenovirus particles produced by the methods of Gotten et al (1992) Proc. Natl. Acad. ScL USA 89, 6094-6098 may also be used. This approach appears to rely on the fact that adenoviruses are adapted to allow release of their DNA from an endosome without passage through the lysosome, and in the presence of, for example transferrin linked to the DNA construct or other genetic construct of the invention, the construct is taken up by the cell by the same route as the adenovirus particle.

This approach has the advantages that there is no need to use complex retroviral constructs; there is no permanent modification of the genome as occurs with retroviral infection; and the targeted expression system is coupled with a targeted delivery system, thus reducing toxicity to other cell types.

It will be appreciated that "naked DNA" and DNA complexed with cationic and neutral lipids may also be useful in introducing the DNA of the invention into cells of the individual to be treated. Non-viral approaches to gene therapy are described in Ledley (1995) Human Gene Therapy 6, 1129-1144. Alternative targeted delivery systems are also known such as the modified adenovirus system described in WO 94/10323 wherein, typically, the DNA is carried within the adenovirus, or adenovirus-like, particle. Michael et al (1995) Gene Therapy 2, 660-668 describes modification of adenovirus to add a cell-selective moiety into a fibre protein. Mutant adenoviruses which replicate selectively in p53-deficient human tumour cells, such as those described in Bischoff et al (1996) Science 274, 373-376 are also useful for delivering the genetic construct of the invention to a cell. Thus, it will be appreciated that a further aspect of the invention provides a virus or virus-like particle comprising a genetic construct of the invention. Other suitable viruses, viral vectors or virus-like particles include lentivirus and lentiviral vectors, HSV, adeno-assisted virus (AAV) and AAV-based vectors, vaccinia and parvovirus.

A fourth aspect of the invention provides a composition comprising a peptide inhibitor of GSK-3 according to the first aspect of the invention or a polynucleotide according to the second aspect of the invention for use in medicine.

A fifth aspect of the invention provides a method of combating a disease or condition mediated by GSK-3 in a patient, the method comprising administering a peptide inhibitor of GSK-3 according to the first aspect of the invention, or a polynucleotide according to the second aspect of the invention, or a pharmaceutical composition according to the third aspect of the invention, to the patient.

Preferably, the patient to be treated is a human. Alternatively, the patient may be an animal, for example a domesticated animal (for example a dog or cat), laboratory animal (for example laboratory rodent, for example mouse, rat or rabbit) or animal important in agriculture (i.e. livestock), for example horses, cattle, sheep or goats.

A sixth aspect of the invention provides the use of a peptide inhibitor of GSK-3 according to the first aspect of the invention, or a polynucleotide according to the second aspect of the invention, in the manufacture of a medicament for combating a disease or condition mediated by GSK-3 activity.

A seventh aspect of the invention provides a peptide inhibitor of GSK-3 according to the first aspect of the invention, or a polynucleotide according to the second aspect of the invention, for use in combating a disease or condition mediated by GSK-3. By a "disease or condition mediated by GSK3 activity" we mean any biological or medical condition or disorder in which effective GSK-3 activity is identified, whether at normal or abnormal levels. The condition or disorder may be caused by the GSK-3 activity or may simply be characterised by GSK-3 activity. That the condition is mediated by GSK-3 activity means that some aspect of the condition can be traced to the GSK-3 activity. It is expected that inhibiting the GSK-3 activity will then prevent, ameliorate or treat the condition so characterised.

Thus, by combating a particular disease or condition we include the meaning of reducing or alleviating symptoms in a patient (i.e. palliative use), preventing symptoms from worsening or progressing, treating the disorder (e.g. by inhibition or elimination of the causative agent), or prevention of the condition or disorder in a subject who is free therefrom.

For example, treatment of type 2 diabetes means alleviating, ameliorating, inhibiting, reducing, or curing the clinical manifestations of type 2 diabetes, either transiently or permanently, including slowing the rate of glucose uptake. Such treatment includes the potentiation of insulin signaling. The "preventing" of type 2 diabetes means inhibiting, delaying, slowing, or preventing the onset of clinical manifestations of type 2 diabetes, either transiently or permanently, including slowing the rate of glucose uptake. Such prevention includes potentiation of insulin signaling. Treatment of a neurodegenerative disorder such as Alzheimer's disease may halt or retard the progression of the disease (e.g., as measured by a reduction in the rate of dementia). Treatment of an affective disorder such as manic depression or bipolar disorder may alleviate or stop the symptoms of the disorder. Treatment of conditions of ischemic insult, such as cerebral stroke, may prevent, halt or reduce neuronal cell death.

Diseases or conditions mediated by GSK-3 include type Il diabetes, hyperglycemia, hyperlipidemia, obesity, neurodegenerative disorders such as Alzheimer's disease, Huntington's disease, Parkinson's disease and AIDS associated dementia, affective disorders such as bipolar disorder, manic depression and schizophrenia, neurotraumatic injuries such as acute stroke, immune potentiation, baldness or hair loss, atherosclerotic cardiovascular disease, hypertension, polycystic ovary syndrome, ischemia, brain trauma or injury, amyotrophic lateral sclerosis, multiple sclerosis, immunodeficiency and cancer including prostate cancer. Methods of treating diabetes mellitus, for example, using a GSK-3 inhibitor .are described in US 2007/0072791. Diabetes mellitus is a heterogeneous primary disorder of carbohydrate metabolism with multiple aetetiologic factors that generally involve insulin deficiency or insulin resistance or both. Type I, juvenile onset, insulin-dependent diabetes mellitus, is present in patients with little or no endogenous insulin secretory capacity. These patients develop extreme hyperglycemia and are entirely dependent on exogenous insulin therapy for immediate survival. Type II, or adult onset, or non-insulin- dependent diabetes mellitus, occurs in patients who retain some endogenous insulin secretory capacity, but the great majority of them are both insulin deficient and insulin resistant. Approximately 95% of all diabetic patients in the United States have non- insulin dependent, Type Il diabetes mellitus (NIDDM), and, therefore, this is the form of diabetes that accounts for the great majority of medical problems. Insulin resistance is an underlying characteristic feature of NIDDM and this metabolic defect leads to the diabetic syndrome. Insulin resistance can be due to insufficient insulin receptor expression, reduced insulin-binding affinity, or any abnormality at any step along the insulin signaling pathway (US 5,861 ,266).

The peptide inhibitors of the invention can be used to therapeutically treat type 2 diabetes in a patient with type 2 diabetes. A therapeutically effective amount of the inhibitor is administered to the patient, and clinical markers, for example blood sugar levels, are monitored. The peptide inhibitors of the invention can further be used to prevent type 2 diabetes in a subject. A prophylactically effective amount of the inhibitor is administered to the patient, and a clinical marker, for example IRS-1 phosphorylation, is monitored.

Treatment of diabetes is determined by standard medical methods. A goal of diabetes treatment is to bring sugar levels down to as close to normal as is safely possible. Commonly set goals are 80-120 milligrams per. deciliter (mg/dl) before meals and 100- 140 mg/dl at bedtime. A particular physician may set different targets for the patent, depending on other factors, such as how often the patient has low blood sugar reactions. Useful medical tests include tests on the patient's blood and urine to determine blood sugar level, tests for glycated haemoglobin level (HbA-ic; a measure of average blood glucose levels over the past 2-3 months, normal range being 4-6%), tests for cholesterol and fat levels, and tests for urine protein level. Such tests are standard tests known to those of skill in the art (American Diabetes Association, 1998). In a specific embodiment, the cancer to be combated is prostate cancer. Methods of treating prostate cancer using a GSK-3 inhibitor are described in WO 2006/018633. It is appreciated that the peptide inhibitor of GSK-3 can be delivered to the area of the prostate by any means appropriate for localised administration of a drug. For example, a solution of the therapeutic molecule in a cell-permeable form can be injected directly into the prostate tumour or can be delivered by infusion using an infusion pump. The therapeutic molecule also can be incorporated into an implantable device which when placed at the desired site at the prostate permits the therapeutic molecule to be released into the surrounding locus. For metastatic disease, the therapeutic molecule in a cell- permeable form can be into the bloodstream.

The peptide inhibitor of GSK-3 may be administered via a hydrogel material. The hydrogel is non-inflammatory and biodegradable. Many such materials now are known, including those made from natural and synthetic polymers. In a preferred embodiment, the method exploits a hydrogel which is liquid below body temperature but gels to form a shape-retaining semisolid hydrogel at or near body temperature. Preferred hydrogel are polymers of ethylene oxide-propylene oxide repeating units. The properties of the polymer are dependent on the molecular weight of the polymer and the relative percentage of polyethylene oxide and polypropylene oxide in the polymer. Preferred hydrogels contain from about 10% to about 80% by weight ethylene oxide and from about 20% to about 90% by weight propylene oxide. A particularly preferred hydrogel contains about 70% polyethylene oxide and 30% polypropylene oxide. Hydrogels which can be used are available, for example, from BASF Corp., Parsippany, NJ, under the tradename Pluronic^R.

At present, there are no known surface antigens that are specific to the prostate. Prostate specific membrane antigen has a high degree of cross-reactivity with other epithelial cells in other organs. However, once a suitable prostate specific antigen has been identified, the peptide inhibitor of GSK-3 may be targeted to the required site using a targeting moiety which binds to or lodges at the site of the prostate cancer. For example, the prostate could be targeted using a prostate-specific antibody with a cleavable linker to a GSK-3 inhibitor. A combined targeting/prodrug approach may be useful.

The peptide inhibitor of GSK-3, or a formulation thereof, may be administered by any conventional method including oral and parenteral (e.g. subcutaneous or intramuscular) injection. Preferred routes include oral, intranasal or intramuscular injection. Routes already known for GSK-3 inhibitors may be used, though it will be appreciated that different localised treatment routes may be more appropriate in combating prostate cancer than for when treating (for example) diabetes. The treatment may consist of a single dose or a plurality of doses over a period of time.

An eighth aspect of the invention provides a method of inhibiting the kinase activity of GSK-3, the method comprising contacting GSK-3 with a peptide inhibitor of GSK-3 as defined in the first aspect of the invention. Alternatively the method may comprise contacting a cell that expresses GSK-3 with a polynucleotide of the second aspect of the invention. The peptide inhibitors may be used to inhibit the activity of GSK-3 in a cell, or in a cell-free assay. Cells are contacted with a peptide inhibitor in an amount effective to inhibit GSK-activity in vitro. The GSK-3 to be inhibited may be GSK-3α or GSK-3β. The method may be carried out in vivo or in vitro.

A ninth aspect of the invention provides a method of identifying a GSK-3 inhibitor, or a lead compound for the identification of a GSK-3 inhibitor, the method comprising: providing a candidate compound which is a peptide as defined in the first aspect of the invention; providing GSK-3, or a fragment thereof; and contacting the GSK-3 or fragment thereof with the candidate compound under conditions permitting at least one activity or function of the GSK-3 or fragment thereof; and determining whether the candidate compound inhibits the at least one activity or function of the GSK-3 or fragment thereof.

By a fragment of GSK-3 we include truncated forms of GSK-3 that retain the kinase activity of GSK-3 or have enhanced kinase activity. For example, Wang et a/ (2004) have described truncated forms of GSK-3 that are more active, and suitable GSK-3 fragments can be generated by calpain-mediated proteolysis (Goήi-Oliver et ai, JBC 2007, in press).

It is appreciated that in this aspect of the invention, providing a candidate compound may comprise providing a polynucleotide of the second aspect of the invention, and the method is performed under conditions allowing the peptide inhibitor to be expressed from the polynucleotide. In this embodiment, the method is typically a cell-based method and determining whether the expressed peptide inhibits the at least one function or activity of the GSK-3 comprises a cell-based assay. GSK-3 is unusual as it is constitutiveiy active and is inhibited in response to upstream signals. Kinase activity is thought to be increased by intramolecular phosphorylation of a tyrosine in the activation loop (Y216 in GSK-3β), whose timing and mechanism is undefined. Lochhead ef a/ (2006) have shown that GSK-3β autophosphorylates Y216 as a chaperone-dependent transitional intermediate possessing intramolecular tyrosine kinase activity and displaying different sensitivity to small-molecule inhibitors compared to mature GSK-3β. After autophosphorylation, mature GSK-3β is then an intermolecular serine/threonine kinase no longer requiring a chaperone.

The peptide inhibitors of the first aspect of the invention have a similar sequence to the GSK-3 tyrosine autophosphorylation site (G EPNVS YICSTYYRAPELI; SEQ ID No: 9), and prevent GSK-3 tyrosine autophosphorylation thus inhibiting GSK-3 activity. Therefore, without wishing to be bound by theory, the inventor considers that a compound that binds to or interacts with the GSK-3 tyrosine autophosphorylation site may be a GSK-3 inhibitor, or a lead compound for the identification of a GSK-3 inhibitor.

Thus, a tenth aspect of the invention provides a method of identifying a GSK-3 inhibitor, or a lead compound for the identification of a GSK-3 inhibitor, the method comprising: providing a candidate compound; providing a polypeptide comprising or consisting of the minimal autophosphorylation site of GSK-3 (EPNVSYICSTYYRAP; SEQ ID No: 10); contacting the polypeptide with the candidate compound; and determining whether the candidate compound binds to or interacts with the polypeptide at the autophosphorylation site; wherein an agent that binds to or interacts with the autophosphorylation site of GSK-3 is a GSK-3 inhibitor, or a lead compound for the identification of a GSK-3 inhibitor.

In an embodiment the polypeptide comprises or consists of the amino acid sequence GEPNVSYICSTYYRAPELI (SEQ ID NO: 9), which is the (non-minimal) autophosphorylation site of GSK-3.

The capability of the candidate compound to bind to or interact with the autophosphorylation site of GSK-3 may be measured by any method of detecting/measuring a protein/protein interaction or other compound/protein interaction, as discussed further below. Suitable methods include methods such as, for example, yeast two-hybrid interactions, co-purification, ELISA, co-immunoprecipitation and surface plasmon resonance methods. Thus, the candidate compound may be considered capable of binding to or interacting with the autophosphorylation site of GSK-3 if an interaction may be detected between the candidate compound and the autophosphorylation site of GSK-3 by ELISA, co-immunoprecipitation or surface plasmon resonance methods or by a yeast two-hybrid interaction or copurification method. It is preferred that the interaction can be detected using a surface plasmon resonance method. Surface plasmon resonance methods are well known to those skilled in the art. Techniques are described in, for example, O'Shannessy DJ (1994) "Determination of kinetic rate and equilibrium binding constants for macromolecular interactions: a critique of the surface plasmon resonance literature" Curr Opin Biotechnol. 5(1):65-71 ; Fivash et a/ (1998) "BIAcore for macromolecular interaction." Curr Opin Biotechnol. 9(1):97-101 ; Malmqvist (1999) "BIACORE: an affinity biosensor system for characterization ofbiomolecular interactions." Biochem Soc Trans. 27(2):335-40.

It is appreciated that screening assays which are capable of high throughput operation are particularly preferred. Examples may include cell based assays and protein-protein binding assays. An SPA-based (Scintillation Proximity Assay; Amersham International) system may be used. For example, an assay for identifying a compound capable of modulating the activity of a protein kinase may be performed as follows. Beads comprising scintillant and a substrate polypeptide that may be phosphorylated may be prepared. The beads may be mixed with a sample comprising the protein kinase and ³²P-ATP or ³³P-ATP and with the test compound. Conveniently this is done in a multi- well (e.g., 96 or 384) format. The plate is then counted using a suitable scintillation counter, using known parameters for ³²P or ³³P SPA assays. Only ³²P or ³³P that is in proximity to the scintillant, i.e. only that bound to the polypeptide, is detected. Variants of such an assay, for example in which the polypeptide is immobilised on the scintillant beads via binding to an antibody or antibody fragment, may also be used.

Other methods of detecting polypeptide/polypeptide interactions include ultrafiltration with ion spray mass spectroscopy/HPLC methods or other physical and analytical methods. Fluorescence Energy Resonance Transfer (FRET) methods, for example, well known to those skilled in the art, may be used, in which binding of two fluorescent labelled entities may be measured by measuring the interaction of the fluorescent labels when in close proximity to each other. A further method of identifying a compound that is capable of binding to the polypeptide is one where the polypeptide is exposed to the compound and any binding of the compound to the said polypeptide is detected and/or measured. The binding constant for the binding of the compound to the polypeptide may be determined. Suitable methods for detecting and/or measuring (quantifying) the binding of a compound to a polypeptide are well known to those skilled in the art and may be performed, for example, using a method capable of high throughput operation, for example a chip-based method. Technology, called VLSIPS™, has enabled the production of extremely small chips that contain hundreds of thousands or more of different molecular probes. These biological chips or arrays have probes arranged in arrays, each probe assigned a specific location. Biological chips have been produced in which each location has a scale of, for example, ten microns. The chips can be used to determine whether target molecules interact with any of the probes on the chip. After exposing the array to target molecules under selected test conditions, scanning devices can examine each location in the array and determine whether a target molecule has interacted with the probe at that location.

It is appreciated that the identification of an agent that binds to or interacts with the autophosphorylation site of GSK-3 may be an initial step in the drug screening pathway, and the identified compounds may be further selected e.g. for the ability to inhibit a kinase activity of GSK-3 inhibitor.

It is appreciated that this method may be a drug screening method, a term well known to those skilled in the art, and the candidate compound may be a drug-like compound or lead compound for the development of a drug-like compound.

The term "drug-like compound" is well known to those skilled in the art, and may include the meaning of a compound that has characteristics that may make it suitable for use in medicine, for example as the active ingredient in a medicament. Thus, for example, a drug-like compound may be a molecule that may be synthesised by the techniques of organic chemistry, less preferably by techniques of molecular biology or biochemistry, and is preferably a small molecule, which may be of less than 5000 Daltons and which may be water-soluble. A drug-like compound may additionally exhibit features of selective interaction with a particular protein or proteins and be bioavailable and/or able to penetrate target cellular membranes or the blood:brain barrier, but it will be appreciated that these features are not essential. The term "lead compound" is similarly well known to those skilled in the art, and may include the meaning that the compound, whilst not itself suitable for use as a drug (for example because it is only weakly potent against its intended target, non-selective in its action, unstable, poorly soluble, difficult to synthesise or has poor bioavailability) may provide a starting-point for the design of other compounds that may have more desirable characteristics.

An eleventh aspect of the invention provides a method of selecting or designing a GSK-3 inhibitor, or a lead compound for the identification of a GSK-3 inhibitor, the method comprising the step of providing a three-dimensional structure of a peptide comprising or consisting of the amino acid sequence PYYVNSGYA (SEQ ID No: 7) that is not phosphorylated at Yj. providing a three-dimensional structure of a peptide comprising or consisting of the amino acid sequence PYYVNSGYA (SEQ ID No: 123) that is phosphorylated at Y₁ using molecular modelling means to select or design a compound that has a three-dimensional structure which is more similar to the three-dimensional structure of the unphosphorylated peptide than of the phosphorylated peptide, thereby to identify a GSK-3 inhibitor, or a lead compound for the identification of a GSK-3 inhibitor.

Preferably, apart from the phosphorylation of the tyrosine residues, the unphosphorylated and phosphorylated peptides are otherwise identical.

The three-dimensional structures of the peptide, whether phosphorylated or unphosphorylated, may be provided by crystallography, or by computer modelling based on a known structure using methods well known in the art.

This aspect of the invention provides a method of selecting or designing a GSK-3 inhibitor, or a lead compound for the identification of a GSK-3 inhibitor, the method comprising the step of: providing a comparison of the three-dimensional structure of a peptide comprising or consisting of the amino acid sequence PYYVNSGYA (SEQ ID No: 7) that is not phosphorylated at Y and of a peptide comprising or consisting of the amino acid sequence PYYVNSGYA (SEQ ID No: 123) that is phosphorylated at Yj. using molecular modelling means to select or design a compound that has a three-dimensional structure which is more similar to the three-dimensional structure of the unphosphorylated peptide than of the phosphorylated peptide, thereby to identify a GSK-3 inhibitor, or a lead compound for the identification of a GSK-3 inhibitor. Preferably, the comparison is of the three-dimensional structure of a peptide comprising or consisting of the amino acid sequence PYYVNSGYA (SEQ ID No: 7) that is not phosphorylated at Y and an otherwise identical peptide that is phosphorylated at Y₁

In an embodiment, the peptide may comprise or consist of the amino acid sequence EPVNPYYVNSGYALAP (SEQ ID NO: 6) or the amino acid sequence REPVNPYYVNSGYALAPATS (SEQ ID NO: 5).

In other words, this aspect of the present invention relates to a computer-assisted method of structure-based drug design of GSK-3 inhibitors based upon the difference between the structures of phosphorylated and unphosphorylated PYYVNSGYA (SEQ ID Nos: 123 and 7 respectively). Suitable methods and computer programmes useful in performing these methods are well known in the art and are described, for example, in US 2007/0072791 and WO 2006/054298.

The three-dimensional structure of GSK-3 is known and includes structural details around the GSK-3 autophosphorylation site in the context of the active and inactive kinase as well as the structure of GSK-3 bound to a peptide from Axin (residues 383- 401) (Bax et al., 2001 , Dajani et al., 2001 , 2003). Therefore, the method may include using molecular modelling means to determine whether the selected or designed compound may bind to or interact with the GSK-3 autophosphorylation site.

The three-dimensional structures may be displayed by a computer in a two-dimensional form, for example on a computer screen. The comparison may be performed using such two-dimensional displays.

The following relate to molecular modelling techniques: Blundell et al (1996) Stucture- based drug design Nature 384, 23-26; Bohm (1996) Computational tools for structure- based ligand design Prog Biophys MoI Biol 66(3), 197-210; Walters ef al (1998) Drug Discovery Today, 3(4), 160-178; Cohen et al (1990) J Med Chem 33, 883-894; Navia et al (1992) Curr Opin Struct Biol 2, 202-210; and Abagyan & Totrov (2001) Curr. Opin. Chem. Biol. 5, 375-382.

The following computer programs, for example, may be useful in carrying out the method of this aspect of the invention: GRID (Goodford (1985) J Med Chem 28, 849-857; available from Oxford University, Oxford, UK); MCSS (Miranker ef a/ (1991) Proteins: Structure, Function and Genetics 11, 29-34; available from Molecular Simulations, Burlington, MA); AUTODOCK (Goodsell et al (1990) Proteins: Structure, Function and Genetics 8, 195-202; available from Scripps Research Institute, La JoIIa, CA); DOCK (Kuntz et a/ (1982) J MoI Biol 161 , 269-288; available from the University of California, San Francisco, CA); LUDI (Bohm (1992) J Comp Aid Molec Design 6, 61-78; available from Biosym Technologies, San Diego, CA); LEGEND (Nishibata ef a/ (1991) Tetrahedron 47, 8985; available from Molecular Simulations, Burlington, MA); LeapFrog (available from Tripos Associates, St Louis, MO); Gaussian 92, for example revision C (MJ Frisch, Gaussian, Inc., Pittsburgh, PA ©1992); AMBER, version 4.0 (PA Kollman, University of California at San Francisco, ©1994); QUANTA/CHARMM (Molecular Simulations, Inc., Burlington, MA ©1994); and Insight Il/Discover (Biosym Technologies Inc., San Diego, CA ©1994). Programs may be run on, for example, a Silicon Graphics™ workstation, Indigo²™ or IBM RISC/6000™ workstation model 550.

Several in silico methods could be employed, for example, via a substructure search for new ligands using programmes such as CHEM DRAW or CHEM FINDER. The basic structure of the unphosphorylated peptide PYYVNSGYA (SEQ ID No: 7), EPVNPYYVNSGYALAP (SEQ ID NO: 6) or REPVNPYYVNSGYALAPATS (SEQ ID No: 5) is taken (or predicted) and various structural features of it (for example the hydrophobic and negatively charged entities) are submitted to a programme which will search a set of chemical company catalogues for chemicals containing this substructure.

These compounds are then screened by eye for groups that are predicted not to be GSK-3 inhibitors, e.g. because they contain substructures that mimic the phosphotyrosine residues, and those are discarded. The remaining chemicals are submitted to a PRODRG server and topologies/co-ordinates for these chemicals are created. These chemicals are modelled into the structure, from which chemicals that are structurally more similar to the structure of the unphosphorylated peptide than the phosphorylated peptide, are selected. Further details of the PRODRG programme are available at http://davapc1.bioch.dundee.ac.uk/programs/prodrg/prodrg. html.

The selected compounds may then be ordered or synthesised and assessed, for one or more of ability to bind to and/or modulate PTPL1 activity. The compounds may be crystallised with the PTPL1 polypeptide and the structure of any complex determined.

The method of the invention may further comprise the steps of providing, synthesising, purifying and/or formulating a compound selected using computer modelling, as described above; and of assessing whether the compound modulates the activity of PTPL1. The compound may be formulated for pharmaceutical use, for example for use in in vivo trials in animals or humans.

It is appreciated that the methods of the tenth and eleventh aspects of the invention may further comprise the step of determining whether the identified candidate compound inhibits at least one activity or function of the GSK-3.

In a preferred embodiment of the ninth, tenth and eleventh aspects of the invention the identified compound is modified, and the modified compound is tested for inhibition of at least one activity or function of GSK-3.

Typically, the at least one activity or function of the GSK-3 is tyrosine kinase activity (e.g. tyrosine autophosphorylation of GSK-3 or tyrosine-phosphorylation of Axin) as described above with aspect to the first aspect of the invention. Methods for measuring GSK-3 tyrosine kinase activity include an in vitro tyrosine autophosphorylation assay in which recombinant purified GSK-3 made using a baculovirus vector to infect Sf9 cells (Dajani ef a/, 2001 ) autophosphorylates on tyrosine in vitro. Other assays include transcription/translation of a GSK3 plasmid expression vector in rabbit reticulocyte lysate, in which tyrosine autophosphorylation occurs during translation (based on Lochhead, 2006). In addition, inhibition of tyrosine phosphorylation of Axin by GSK-3 can be measured in a cell-based assay in which Axin and GSK-3 are co-expressed in cells (e.g. COS cells) and anti-phosphotyrosine western blots of cell extracts are used to detect the level of tyrosine-phosphorylated Axin.

Alternatively or additionally, the activity or function of the GSK-3 may be a serine kinase activity which can be measured by an in vitro serine kinase assay e.g. phosphorylation of a phosphopeptide comprising the GSK3 phosphorylation sites in glycogen synthase (Ryves ef a/., 1998) or other substrates such as elF2B. Further alternatively or additionally, the activity or function of the GSK-3 may be inhibition of prostate cancer cell proliferation which can be measured by a cell-based assay for inhibition of 22Rv1 prostate cancer cell proliferation (Mazor ef a/., 2004).

The inhibitor of GSK-3 may be selective for GSK-3α or GSK-3β. Thus, in order to determine the selectivity of the peptide inhibitor for GSK-3α or GSK-3β, the screening aspects of the invention may comprise testing the identified compound or the modified compound for inhibition of GSK-3α and GSK-3β kinase activity. It is preferred if the inhibitor of GSK-3 is selective for GSK-3 as defined above with respect to the first aspect of the invention. Thus, in order to determine the selectivity of the peptide inhibitor of GSK-3, the screening aspects of the invention preferably include the subsequent step of testing the identified compound or the modified compound for inhibition of kinase activity of at least one human protein kinase other than GSK-3, as discussed above.

Preferably, the method includes testing the identified compound or the modified compound for inhibition at least 2 or 3 or 4 or 5 or at least 10 other human protein kinases. Methods for determining the selectivity of a GSK-3 inhibitor are described by Ring et al (2003) with respect to 20 different protein kinases, and the at least one other protein kinase may be any one or more of them.

It is preferred if the identified compound or the modified compound has an IC50 value for GSK-3 at least ten times lower than for human CDC2, one of the most closely related kinases, and preferably at least 100, or 500 times lower. More preferably, the identified compound or the modified compound is a GSK-3 selective inhibitor having an IC50 value more than 1 ,000 or 5,000 times lower for GSK-3 than for human CDC2. Most preferably, the identified compound or the modified compound is a GSK-3 selective inhibitor having an IC₅₀ value at least five times lower than for all other human protein kinases, and preferably at least 10, 50, 100 or 500 times lower.

The screening methods preferably also comprise the further step of testing the identified compound or the modified compound for efficacy in a model of a disease or condition mediated by GSK-3. The model may be a cellular model or an animal model. The model may be a model for any of the diseases or condition mediated by GSK-3 mentioned above.

Suitable models include Tet/GSK-3beta transgenic mice as a model for Alzheimer's disease (Lucas et al., EMBO J. 2001) and rat diabetes models (see Ring et al., 2003).

The screening methods may further comprise the step of formulating the compound identified into a pharmaceutically acceptable composition.

Compounds may also be subjected to other tests, for example toxicology or metabolism tests, as is well known to those skilled in the art. Thus, the invention includes a method of making a pharmaceutical composition comprising the step of mixing the compound identified using the methods described above with a pharmaceutically acceptable carrier.

All of the documents referred to herein are incorporated herein, in their entirety, by reference.

The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

The invention is now described in more detail by reference to the following, non-limiting, Figures and Examples.

Brief Description of the Drawings

FIGURE 7. Phosphorylation ofAxin by GSK3. Illustration depicting known phosphorylation sites in Axin.

FIGURE 2. Expression of GSK-3β induces Axin tyrosine phosphorylation.

9E10 (anti-myc tag) immune precipitates from COS cells transfected with plasmids encoding myc-Axin (lanes 1 to 4) and kinase-dead GSK-3β (lane 1), GSK-3β (lane 2), FYN (lane 3) and FAK (lane 4), were probed with RC20H anti-phosphotyrosine antibodies. Arrows indicate the positions of tyrosine-phosphorylated Axin and GSK-3β.

FIGURE 3. Tyrosine phosphorylation of Axin is inhibited by lithium.

(A) Anti-GFP immune precipitates from COS cells transfected with plasmids encoding GFP-Axin (lanes 1 to 6) and GSK-3β (lanes 1 to 3) or FYN (lanes 3 to 6). After transfection cells were treated with 10 μM PP2 (lanes 2 and 5) or 20 mM lithium chloride (lanes 3 and 6). Immune precipitates were probed with RC20H anti-phosphotyrosine antibodies (upper panel). The blot was then stripped and reprobed with anti-GFP mAb (lower panel).

(B) COS cells were transfected with plasmids encoding GFP-Axin and GSK-3β. After transfection cells were treated with DMSO carrier (control) (lane 1 ) or GSK-3 inhibitors SB216763 (5 micromolar, Sigma) (lane 2), Azakenpaullone (2 micromolar, Merck) (lane 3) or Inhibitor X (5 micromolar, Merck) (lane 4) for 3 hours. Cell extracts were then probed with RC20H anti-phosphotyrosine (upper panel) or anti-GFP (lower panel). The arrow indicates the position of GFP-Axin.

FIGURE 4. GSK-3a phosphorylates Axini on tyrosine COS cells were transfected with plasmids encoding myc-epitope-tagged Axin and empty vector (V), FYN, GSK-3α or GSK-3β. After 24 hours, cell extracts were probed with 9E10 (top left, to detect Axin), anti-GSK3 (middle, left), or RC20H anti-phosphotyrosine (bottom left). In addition, 9E10 immune-precipitates were probed with RC20H (right). The arrows indicate the position of tyrosine-phosphorylated Axin.

FIGURE 5. GSK-3α and GSK-3β phosphorylate Axin2 on tyrosine COS cells were transfected with plasmids encoding myc-epitope-tagged Axin or myc- epitope-tagged Axin2 and empty vector (V), FYN, GSK-3α or GSK-3β. After 24 hours, cell extracts were probed with 9E10 (left, to detect Axin and Axin2). In addition, 9E10 immune-precipitates of myc-epitope-tagged Axin2 were probed with RC20H (right, top) and reprobed with 9E10 (right, bottom). The arrow indicates the position of tyrosine- phosphorylated Axin2.

FIGURE 6. Tyrosine phosphorylation of Axin by GSK~3β requires an intact GSK-3 binding site.

Anti-GFP immune precipitates from COS cells transfected with plasmids encoding GFP- Axin (lanes 1 to 3) or GFP-Axin L397P (lanes 4 to 6) with empty vector (lanes 1 and 4), GSK-3β (lanes 2 and 5) or FYN (lanes 3 and 6). Immune precipitates were probed with RC20H anti-phosphotyrosine antibodies. The positions of GFP-Axin (left arrow) and GFP-Axin L397P (right arrow) are indicated.

FIGURE 7. Recombinant GSK-3β phosphorylates Axin on tyrosine residues in vitro.

A, anti-GFP immune precipitates from COS cells transfected with GFP-Axin were incubated for 5 min (lanes 1 and 3) or 40 min (lanes 2 and 4) with recombinant GSK-3β in the presence of kinase buffer with 10 mM NaCI (lanes 1 and 2) or 10 mM LiCI (lanes 3 and 4) and probed with anti-phosphotyrosine antibodies. The position of GFP-Axin is indicated with an arrow. B, 9E10 immune precipitates from COS cells transfected with myc-Axin were untreated (lanes 1 and 2) or heat-inactivated (lanes 3 and 4), incubated in kinase buffer alone (lanes 1 and 3) or with recombinant GSK-3β (lanes 2 and 4) and then probed with anti-phosphotyrosine antibodies. The position of myc-Axin is indicated with an arrow. C, MBP-Axin 298-506 (lanes 1 to 4) or MBP-Axin (lanes 5 to 8) were incubated for 0 min (lanes 1 and 5), 5 min (lanes 2 and 6) or 30 min (lanes 3 and 7) with recombinant GSK-3β in kinase buffer with ATP. Kinase assays were also performed for 30 min without GSK-3β (lanes 4 and 8). Samples were probed with anti-phosphotyrosine antibodies. The positions of MBP-Axin 298-506 (left arrow) and MBP-Axin (right arrow) are indicated. The phosphorylation of MBP-Axin was weaker than that of MBP-Axin 298- 506 because there was less of this substrate present in the assay. D, myc-Axin was incubated in kinase buffer with recombinant GSK-3β and radiolabeled ATP and then subjected to phosphoamino acid analysis. The positions of phosphoserine (PS), phosphothreonine (PT), phosphotyrosine (PY) and phosphopeptides are indicated.

FIGURE 8. GSK-3β phosphorylates Axin at two major tyrosine sites in vivo.-

Anti-GFP immune precipitates from COS cells transfected with plasmids encoding GFP- Axin (lanes 1 to 3), GFP-Axin Y309F (lanes 4 to 6), GFP-Axin Y315F (lanes 7 to 9), GFP-Axin Y309F/Y315F (lanes 10 to 12) with empty vector (lanes 1 , 4, 7 and 10), GSK- 3β (lanes 2, 5, 8 and 11) or FYN (lanes 3, 6, 9 and 12). Immune precipitates were probed with RC20H anti-phosphotyrosine antibodies (upper panels). The blots were then stripped and reprobed with anti-GFP mAb (lower panels).

FIGURE 9. A peptide containing Axin residues Y309 and Y315 inhibits GSK-3β activity. A, Alignment of residues REPVNPYYVNSGYALAPATS (AxPEP) (SEQ ID No: 5) surrounding the Axin tyrosine phosphorylation sites with the GSK-3β activation loop. In large-type font are Axin tyrosine phosphorylation sites Y309 and Y315 and the Axin serine phosphorylation site S322 (Yamamoto et al 2001), the GSK-3β tyrosine phosphorylation site Y216 and a second tyrosine in GSK-3β that is phosphorylated by ZAK kinase (Kim et al 2002). In bold are residues common to Axin and GSK-3β. In italics are residues in AxPEP and the GSK-3β activation loop that may also be homologous. S, kinase assays were conducted using purified MBP-Axin-298-506 and purified recombinant (Sf9) GSK-3β for 5 min (lanes 1 , 3 and 5) or 30 min (lanes 2, 4 and 6) in the absence of peptide (lanes 1 and 2), with AxPEP (lanes 3 and 4) or with GSM peptide (lanes 5 and 6). Samples were probed using anti-phosphotyrosine antibodies. The positions of MBP-Axin-298-506 (right arrow) and GSK-3β(left arrow) are indicated. C, kinase assays were conducted using purified MBP-Axin-298-506 and purified (Sf9) recombinant GSK-3β for 30 min in the absence of peptide (lane 1), with AxPEP (PEP; lanes 2 and 4) or with AxPEPP (PEPP; lanes 3 and 5). The samples in lanes 4 and 5 were pretreated with the tyrosine phosphatase, TC-PTP. All samples were subsequently heat-inactivated prior to addition of GSK-3β. Samples were probed using anti- phosphotyrosine antibodies. The positions of MBP-Axin-298-506 (right arrow) and GSK- 3β (left arrow) are indicated. Example 1 :Tyrosine phosphorylation of Axin by glycogen synthase kinase-3

Summary Axin negatively regulates Wnt signaling by facilitating the phosphorylation of β-catenin by casein kinase-l (CKI) and glycogen synthase kinase-3β (GSK-3β, marking it for proteosomal degradation. In addition, serine and threonine phosphorylation of Axin by GSK-3β increases its stability and its affinity for β-catenin. Here, we present evidence that Axin undergoes GSK-3β-dependent tyrosine phosphorylation. Axin tyrosine phosphorylation is blocked by several GSK-3 inhibitors, but not by PP2, a tyrosine kinase inhibitor. A point-mutant of Axin that cannot bind GSK-3β is not phosphorylated on tyrosine. In addition, GSK-3β phosphorylates Axin2 on tyrosine and GSK-3α phosphorylates both Axin and Axin2 on tyrosine. Purified recombinant GSK-3β phosphorylates purified Axin on tyrosine in vitro, suggesting that tyrosine phosphorylation in vivo is direct. Deletion and point mutation analyses suggest that Y309 and Y315 are the in vivo tyrosine phosphorylation sites. These residues lie in a sequence resembling the GSK-3β activation loop containing the GSK-3β autophosphorylation site. An unphosphorylated peptide containing the Axin tyrosine phosphorylation sites inhibits GSK-3β tyrosine kinase activity in vitro, while a tyrosine-phosphorylated form of this peptide does not. These observations are consistent with a model in which a GSK-3β pseudo-activation loop in Axin inhibits GSK-3β and tyrosine phosphorylation of Axin relieves this inhibition.

Introduction In the current model of the Wnt signaling pathway, the cytosolic level of β-catenin is controlled by phosphorylation and ubiquitin-dependent degradation. Wnt signals stabilise β-catenin, allowing it to associate with Tcf/LEF-1 family members and enter the nucleus to regulate gene expression (1 ,2). By directly associating with β-catenin, GSK- 3β and CKI, Axin (3) and the related protein Axin2 (conductin/axil) (4,5) facilitate β- catenin phosphorylation (6-15). Overexpression of Axin destabilises β-catenin and blocks axis duplication by XWnt-8 in Xenopus embryos (3,5) and Wnt-induced accumulation of β-catenin in cell culture (13).

Axin itself is a substrate for GSK-3β (7). Phosphorylation of rat Axin (Axin) on serine residue 322 promotes its stability (12,16) , while phosphorylation of human Axin on threonine 609 and serine 614 (equivalent to threonine 485 and serine 490 in Axin) by either GSK-3β or by CDK2 promotes association with β-catenin (17,18). Moreover, Wnt1 and Wnt3A induce Axin dephosphorylation and reduce the affinity of β-catenin for Axin (19). These observations support a model in which Wnt signals lead to dephosphorylation of Axin, resulting in the release of β-catenin from the Axin complex and the degradation of Axin. This likely involves the Wnt receptors LRP-5/-6, which recruit Axin to the membrane and promote its degradation (20,21). Wnt signaling requires phosphorylation of a PPPSP (SEQ ID No: 124) motif in LRP5/6, which creates an inducible docking site for Axin (22). Kinases involved in this phosphorylation were recently identified and include CKl family members and GSK-3 (23-26).

GSK-3β is a serine/threonine kinase that phosphorylates substrates either with the sequence S/TP or S/TXXXS/T (SEQ ID No. 125), where the second serine/threonine residue has been phosphorylated by another kinase (27). There is some evidence to suggest that GSK-3β is also a tyrosine kinase. Purified GSK-3β autophosphorylates on tyrosine residues in vitro (28). The autophosphorylation site (Y216) is in the activation loop, and phosphorylation at this site activates the kinase (29). Other kinases have also been reported to phosphorylate GSK-3β on tyrosine, for example ZAK (30). However, when rabbit GSK-3β is expressed and purified from E. coli, it is already tyrosine- phosphorylated (28), indicating that mammalian GSK-3β is capable of tyrosine autophosphorylation. Indeed, tyrosine phosphorylation of GSK-3β has been shown to be an intramolecular autophosphorylation event (31). Other GSK-3 family members have similar properties (32-34). It is unclear if mammalian GSK-3β can phosphorylate other proteins on tyrosine, although this was recently reported for the yeast GSK-3β homolog, Rim11 (35). Here we present evidence that GSK-3β phosphorylates Axin on two tyrosine residues in a sequence that resembles the autophosphorylation site in GSK-3β.

Experimental procedures

DNA Plasmid Constructs

Plasmids encoding myc-tagged Xenopus β-catenin (36) and myc-tagged rat Axin (7) have been described. Myc-tagged activated (S9A mutant) human GSK-3β (37) was provided by Virginia Lee (University of Pennsylvania School of Medicine, Philadelphia, PA). The kinase-inactive mutant of GSK-3β (K85G) was generated by oligonucleotide site-directed mutagenesis of S9A GSK-3β. Both forms of GSK-3β were subcloned into pMT23 using EcoRI/Notl. pcDNA plasmids encoding wild-type GSK-3α and GSK3β were from Richard Killick (Kings College London) and Trevor Dale (Cardiff University), respectively. pSG5 Fyn was from Sara Courtneidge (The Burnham Institute, La JoIIa, CA). FAK plasmid was from Jun-Lin Guan (Cornell University, Ithaca, NY). Wnt-1 was from Tony Brown (Cornell University, New York, NY) and was subcloned into pMT23 using EcoRI. HA-tagged Tcf-4 plasmid was from Marc van de Wetering and Hans Clevers (Hubrecht Laboratory, Utrecht, The Netherlands). The OT Tcf-responsive reporter DNA (38) was provided by Ken Kinzler and Bert Vogelstein (Johns Hopkins Oncology Center, Baltimore, MD).

Unless stated otherwise, Axin and Axin deletion mutants used for GFP fusions are as previously described (7). GFP-Axin was generated by inserting the Smal/EcoRV fragment from pBSKS Axin into the Smal site of pEGFP-C1. For GFP-Axin 1-229, the EcoRI/BamHI fragment from myc-Axin 1-229 was inserted into pEGFP-C2. For GFP- Axin 1-353, the EcoRI/BamHI fragment from myc-Axin 1-353 (40) was inserted into pEGFP-C2 cut with EcoRI/BamHI. For GFP-Axin 298-713, the EcoRI fragment from myc-Axin 298-713 was inserted into pEGFP-C2. For GFP-Axin 298-506, the EcoRI/Pstl fragment from myc-Axin 298-713 encoding residues 298-506 was inserted into pEGFP- C2 cut with EcoRI/Pstl. For GFP-Axin 298-832, the BamHI fragment from myc-Axin encoding residues 354-832 was inserted into GFP-Axin 298-713 from which the BamHI fragment encoding residues 354-713 had been removed. For GFP-Axin 1-713, the BamHI fragment from myc-Axin 298-713 encoding residues 354-713 was inserted into GFP-Axin from which the BamHI fragment encoding residues 354-832 had been removed. For GFP-Axin 713-832, the Pvull/EcoRV fragment from pBSKS Axin encoding these residues was inserted into pEGFP-C2 cut with Smal. For GFP-Axin 508-832, GFP-Axin was cut with Pstl and religated, thereby removing the coding region for Axin residues 1-507. For GFP-Axin 312-832, we made use of an EcoRI site that had been introduced into GFP-Axin Y310F (see below). GFP-Axin Y310F was partially digested with EcoRI and the fragment containing GFP-Axin 312-832 was then religated.

Polymerase chain reaction-based mutagenesis was performed with the QuikChangeTM site-directed mutagenesis kit (Stratagene, La JoIIa, CA) to generate tyrosine to phenylalanine and leucine to proline mutations in GFP-Axin and in GFP-Axin-298-506 (CLONTECH). All mutants were confirmed by sequencing (MWG BIOTECH, GmbH). The oligonucleotides used to generate tyrosine site mutants included a silent EcoRI site at the position encoding N312 (AAT). This site was then used to generate the Y309F/Y315F double mutant.

Myc-epitope-tagged Axin2 corresponds to pEF-BOS-Myc/Axil in Yamamoto et a/. [MoI Cell Biol, May 1998, Vol. 18, No. 5, p. 2867-2875). For the experiment in Figure 5, myc- epitope-tagged Axin plasmid is pEF-BOS-Myc/rAxin (Ikeda et al., 1998). Cell Culture and Transfections

COS, DLD-1 and HEK 293 cells were grown in DMEM and NTERA-2 cells were grown in DMEM/Ham's F12. Media were supplemented with 10% FCS and antibiotics. Transient transfections were done according to manufacturers protocols using LipofectAmine Plus reagent (Invitrogen) or Fugene 6 (Roche) in 6-well tissue-culture plates. For in vivo tyrosine phosphorylation of Axin, COS cells were transfected with empty vector (1 μg), GSK-3 (1 μg) or pSG5 FYN (0.5 μg), together with myc-Axin (0.5 μg) or the various GFP- Axin fusion plasmids (0.3 μg). GSK-3 inhibitors (Sigma and Merck), lithium chloride (20 mM) and PP2 (10 μM in DMSO, Calbiochem) were diluted in DMEM and added to cells for 3 h (GSK-3 inhibitors) or 16 h (lithium chloride and PP2) 16-24 hours after transfection.

Preparation and analysis of extracts

Cells were harvested for preparation of extracts 24 to 36 h after transfection. For western blotting and immune precipitation, cells were washed in PBS and lysed in modified RIPA buffer (36). Immune precipitates were collected using polyclonal anti- GFP (41), monoclonal anti-GFP (Boehringer Mannheim) or 9E10 anti-myc tag antibody (Sigma). Western blots were probed for phosphotyrosine using RC20H (BD Transduction Labs, Lexington, KY), rabbit anti-phosphotyrosine (BD Transduction Labs, Lexington, KY) or 4G10 (UBI₁ Lake Placid, NY), and for myc-tagged proteins using 9E10. Western blots were probed for GFP using polyclonal or monoclonal anti-GFP, followed by pre-adsorbed HRP-conjugated antibodies (Jackson Laboratories, West Grove, PA), and developed using chemiluminescence (Supersignal West Pico, Pierce, Rockford, IL). Prior to re-probing, western blots were stripped according to a protocol from BD Transduction Labs. Chemiluminescent signals on western blots were quantified on a Fluor-S MAX Multilmager (Bio-Rad).

Kinase assays

Washed immune precipitates from COS cells transfected with myc-Axin or GFP-Axin plasmids were washed once in kinase buffer (20 mM Tris pH 7.5, 10 mM MgCl2, 5 mM DTT, 1 μg/ml aprotinin and 1 μg/ml leupeptin) prior to kinase assay. Heat-treatment was at 56⁰C for 10 min. Kinase assays were initiated by addition of 20 μl kinase buffer containing recombinant GSK-3β purified from E. coli (NEB, Beverly, MA) and 200 μM ATP, followed by gentle agitation at 3O⁰C for 30 min, unless specified otherwise. In some experiments, purified MBP-Axin fusion proteins (7) were used as a substrate and the recombinant GSK-3β used was purified from Sf9 cells (43). For phospho-amino acid analysis of myc-Axin, 10 μCi γ-ATP was included in the presence of 100 μM unlabeled ATP. The presence of unlabeled ATP significantly enhanced tyrosine phosphorylation of myc-Axin by GSK-3β, relative to its serine/threonine phosphorylation. Phospho-amino acid analysis was done by transfer of radiolabeled proteins to lmmobilon P (Millipore, Bedford, MA) and following the protocol of Kamps and Sefton (44).

The amounts of each component used in the kinase assays were as follows: GSK-3β (NEB), 2 Units; GSK-3β purified from Sf9 cells (43), 1.2 pmoles; MBP-Axin-298-506, 5 pmoles; MBP-Axin 1 pmoles; peptides 0.2 μmoles. The peptides used were AxPEP, AxPEPP or GSM (45). AxPEP is REPVNPYYVNSGYALAPATS (SEQ ID No: 5), which corresponds to Axin residues 303-322, AxPEPP is the same peptide with both tyrosines phosphorylated. AxPEP and AxPEPP were synthesised and HPLC purified by WIBR support services (University College London). GSM is a GSK-3β substrate based on the phosphorylation site in glycogen synthase. It has the sequence

RRRPASVPPSPSLSRHSSHQRR (SEQ ID NO: 126), where S is phosphoserine. To avoid nonspecific inhibitory effects, all peptides were further purified for kinase assays according to Ryves et a/ (45). Dephosphorylation was done by adding 5 units of T-cell protein tyrosine phosphatase (TC-PTP, NEB). Heat inactivation of TC-PTP was done at 65 ⁰C for 30 min.

Results

Coexpression of Axin and GSK-3β results in Axin tyrosine phosphorylation

In experiments designed to identify substrates of the Axin/GSK-3β complex using anti- phosphoamino acid antibodies, it became apparent that co-expression of GSK-3β with myc-tagged rat Axin (Axin) in COS cells resulted in the tyrosine phosphorylation of the latter (Figure 2, lane 2). Expression of a kinase-dead form of GSK-3β did not induce Axin tyrosine phosphorylation (lane 1). Axin was also tyrosine-phosphorylated when co- expressed with FYN, a member of the Src family of tyrosine kinases (lane 3), but not when co-expressed with the focal adhesion kinase, FAK (lane 4). Tyrosine- phosphorylated GSK-3β was also present in cells transfected with GSK-3β (lane 2), but not in cells expressing kinase-dead GSK-3β (lane 1). Similar results were obtained using other anti-phosphotyrosine antibodies (data not shown). The majority of the subsequent co-transfection experiments were conducted using a GFP-Axin fusion protein rather than myc-Axin, since GSK-3β also has a myc-epitope tag. One possible explanation for GSK-3β-induced tyrosine phosphorylation of Axin is that GSK-3β activates a tyrosine kinase than phosphorylates Axin. A candidate tyrosine kinase is FYN, which has been reported to associate with GSK-3β (46) , and which we have shown induces Axin tyrosine phosphorylation (Figure 2). In order to test this, GFP- Axin was co-expressed with GSK-3β or FYN in the presence of either lithium, an inhibitor of GSK-3β (47,48) or PP2, an inhibitor of SRC family tyrosine kinases (49). GSK-3β- induced tyrosine phosphorylation of GFP-Axin was inhibited by lithium (Figure 3A, upper panel, lane 3) but not by PP2 (lane 2), while FYN-induced tyrosine phosphorylation of GFP-Axin was inhibited by PP2 (lane 5) and, to a lesser extent, by lithium (lane 6).

Axin stability is increased by GSK-3β-dependent serine phosphorylation (12). Therefore, in order to determine if any of the treatments affected the stability of GFP-Axin, the blots were stripped and re-probed with anti-GFP antibodies (lower panel). In this and other experiments (data not shown), GSK-3β expression increased the expression level of GFP-Axin, and this increase was abrogated by lithium. In contrast, expression of FYN reduced the expression level of GFP-Axin.

Since lithium inhibits many enzymes, more specific GSK-3 inhibitors were tested for their effects on GSK-3-induced tyrosine phosphorylation of Axin (Figure 3B). The GSK-3 inhibitors SB216763, Azakenpaullone and Inhibitor X (6-Bromoindirubin-acetoxime) all inhibited GSK-3-induced tyrosine phosphorylation of Axin.

GSK-3α phosphorylates Axin on tyrosine

Since GSK-3α shares many properties with the highly-related kinase GSK-3α the ability of GSK-3α to induce tyrosine phosphorylation of Axin was tested by coexpression in COS cells (Figure 4). Similar to GSK-3α (lane 4), GSK-3α induced tyrosine phosphorylation of Axin (lane 3). Tyrosine phosphorylation of Axin by GSK-3α was detected both in extracts (bottom left panel) and in Axin immune-precipitates (right panel).

GSK-3α and GSK-3B phosphorylate Axin2 on tyrosine

Since Axin and Axin2 are highly related in sequence, the possibility that GSK-3β and GSK-3α also induce tyrosine phosphorylation of Axin2 was tested (Figure 5). The expression level of transfected Axin2 was much lower than for Axin (left panel) and this made it difficult to detect tyrosine phosphorylation of Axin2. However, when larger amounts of extract were used, it was clear that GSK-3α and GSK-3β both increased tyrosine phosphorylation of Axin2 (right, upper panel, lanes 3 and 4 respectively). The effects of FYN expression were less conclusive (lane 1), since expression of FYN greatly reduced Axin2 expression levels (see left panel).

In order to examine further the requirements for GSK-3β-induced Axin tyrosine phosphorylation, a point mutation (L397P) was made in GFP-Axin that abolishes its interaction with GSK-3β (50) but not with other kinases such as CKI (51). GSK-3β did not induce tyrosine phosphorylation of GFP-Axin L397P (Figure 6, lane 5), suggesting that GSK-3β-induced tyrosine phosphorylation of Axin requires a direct interaction between these proteins. In contrast, FYN-dependent tyrosine phosphorylation of GFP- Axin L397P (lane 6) occurred to a similar extent as for GFP-Axin (lane 3). Phosphorylation of GFP-Axin L397P by FYN was also reduced by lithium (data not shown), suggesting that the effects of lithium on FYN-dependent phosphorylation of Axin are independent of GSK-3β.

Recombinant GSK-3β phosphorylates Axin on tyrosine residues in vitro

The experiments above suggest that GSK-3β phosphorylates Axin directly on tyrosine residues. In order to determine whether this was the case, in vitro kinase assays were performed using purified components. GFP-Axin immune precipitates from transfected COS cells were incubated for 5 or 40 min with recombinant GSK-3β in the presence of kinase buffer containing either 10 mM LiCI or 10 mM NaCI as a control, and then probed for the presence of phosphotyrosine. Recombinant GSK3-β phosphorylated GFP-Axin on tyrosine residues (Figure IA, lanes 1 and 2), and this phosphorylation was reduced in the presence of LiCI (lanes 3 and 4). Similar results were obtained using myc-Axin (data not shown).

Under some lysis conditions, GFP-Axin immune precipitates contained an associated kinase activity, possibly endogenous GSK-3β, which phosphorylated GFP-Axin on tyrosine (data not shown). Therefore, in order to ensure that phosphorylation in vitro was mediated by recombinant GSK-3β, experiments were performed after heat-treatment of immune precipitates to inactivate any associated kinases. Recombinant GSK-3β phosphorylated heat-treated myc-Axin on tyrosine (Figure IB, lane 4) to the same extent as untreated myc-Axin (lane 2). In order to rule out the possibility that a heat-stable Axin-associated protein was required for GSK3β-dependent tyrosine phosphorylation, kinase assays were done using purified recombinant MBP-Axin fusion proteins (Figure 5C). Recombinant GSK3-β phosphorylated MBP-Axin 298-506 (see lanes 1 to 3) and MBP-Axin (see lanes 5 to 7) on tyrosine residues in vitro. In order to ensure that the anti-phosphotyrosine antibodies were not cross-reacting with phosphoserine or phosphothreonine epitopes on Axin, in vitro kinase assays were performed in the presence of radiolabeled ATP. Phosphoamino acid analysis of phosphorylated myc-Axin indicated the presence of phosphoserine, phosphothreonine and phosphotyrosine (Figure 7D).

GSK3-3 phosphorylates Axin at two major tyrosine sites in vivo

In order to determine the domains of Axin containing the tyrosine phosphorylation sites, a number of deletion mutants of GFP-Axin were co-expressed with GSK-3β in COS cells and probed for the presence of phosphotyrosine. GSK-3β-dependent tyrosine phosphorylation of the majority of the deletion mutants correlated with the abilities of these mutants to associate with GSK-3β. In addition, this analysis indicated that there were at least two tyrosine phosphorylation sites, a strong site (or sites) in residues 298- 312 and a weaker site (or sites) in residues 312-506.

To identify the phosphorylation sites in Axin, candidate tyrosine residues in GFP-Axin were mutated to phenylalanine. Mutation of Y310 or Y351 did not affect Axin tyrosine phosphorylation (data not shown). In contrast, mutation of either Y309 (Figure 8, upper panel, lane 5) or Y315 (lane 8) significantly reduced tyrosine phosphorylation of GFP- Axin (compare with lane 2). Mutation of both Y309 and Y315 together eliminated GSKβ- dependent tyrosine phosphorylation of GFP-Axin (lane 11). Thus, Y309 and Y315 are the sites in Axin that are phosphorylated by GSK-3β. Mutation of these sites did not significantly affect the expression levels of GFP-Axin (Figure 8, lower panel). Interestingly, mutation at Y309 and Y315 either alone (lanes 6 and 9) or together (lane 12) also significantly reduced FYN-induced tyrosine phosphorylation of GFP-Axin. Mutation of Y310 together with Y309 and Y315 did not further affect tyrosine phosphorylation by GSK-3β or by FYN (data not shown).

A peptide containing Axin residues Y309 and Y315 inhibits GSK-3β activity We noted that Y309 and Y315 of Axin lie in a sequence that resembles the sequence surrounding Y216, the tyrosine phosphorylation site in the activation loop of GSK-3β

(Figure 9/4). A peptide containing the Axin sequence containing Y309 and Y315

(AxPEP) was synthesised for use as a substrate in in vitro kinase assays. However, it was not possible to phosphorylate this peptide in vitro using GSK-3β. In order to determine if this peptide affected GSK-3β kinase activity, GSK-3β kinase assays were done using purified MBP-Axin-298-506 as a substrate. Here we used recombinant GSK-

3β purified from Sf9 cells. Unlike recombinant GSK-3β from NEB, this preparation of GSK-3β is not fully phosphorylated on tyrosine (43), and therefore underwent significant tyrosine autophosphorylation in vitro (Figure 9S, lane 2). Importantly, MBP-Axin-298-506 was also tyrosine-phosphorylated in this assay. Interestingly, AxPEP significantly inhibited both GSK-3β autophosphorylation and Axin tyrosine phosphorylation (lane 4). Inhibitory effects were detectable at 40-fold molar excess of peptide over GSK-3β and complete inhibition required 800-fold molar excess of peptide. In contrast, GSM, a peptide used as a GSK-3β substrate in kinase assays (45), did not significantly affect GSK-3β autophosphorylation or tyrosine phosphorylation of MBP-Axin-298-506 (lane 6). The inhibition of GSK-3β by AxPEP would explain why we could not phosphorylate this peptide using GSK-3β in kinase assays.

In order to determine whether tyrosine phosphorylation of AxPEP affected its ability to inhibit GSK-3β activity, a tyrosine-phosphorylated version of AxPEP, AxPEPP, was synthesised and tested (Figure 9C). In contrast to the inhibitory effect of AxPEP (lane 2), AxPEPP did not affect GSK-3β autophosphorylation or MBP-Axin-298-506 tyrosine phosphorylation (lane 3). Moreover, dephosphorylation of AxPEPP prior to the kinase assay enabled it to inhibit GSK-3β (lane 5). Phosphatase treatment of AxPEP did not affect its inhibitory activity (lane 4). These results suggest that Axin controls GSK-3β activity by inhibiting autophosphorylation, and that this function of Axin is blocked by tyrosine phosphorylation of Axin.

Discussion

In this work we have provided evidence that the serine/threonine kinases GSK-3α and GSK-3β can phosphorylate the associated proteins, Axin and Axin2, on tyrosine residues. This phenomenon appears to be specific to Axin and Axin2, since we do not detect other tyrosine-phosphorylated proteins in extracts of cells expressing GSK-3β and Axin (data not shown). There are very few examples where GSK family kinases phosphorylate other proteins on tyrosine ((33), this work). Tyrosine phosphorylation of substrates by GSK-3 family kinases might only occur in instances where there is a separate, direct interaction between the kinase and its substrate (Figure 5 and Table 1). Table 1 : Summary of data obtained from anti-phosphotyrosine immunoblot analysis of GFP immune precipitates constructs cotransfected with GSK-3β.

Although controversial, there is some precedent for kinases of the GSK family having tyrosine kinase activity. When rabbit GSK-3β is expressed and purified from E. coli, it is already tyrosine-phosphorylated (28), indicating that mammalian GSK-3β is capable of tyrosine autophosphorylation. Moreover, purified GSK-3β autophosphorylates on tyrosine residues in vitro (28). In fission yeast, Skp1 autophosphorylates on tyrosine (32) and, in budding yeast, Rim11 p autophosphorylates on tyrosine (34) and also associates with, and tyrosine phosphorylates lmeip (33). In contrast, a kinase called ZAK tyrosine phosphorylates GSK-3 in Dictyostelium (30). It remains to be determined which process, autophosphorylation or phosphorylation by other kinases, is more important for controlling GSK-3 activity in mammalian cells.

Tyrosine phosphorylation of Axin by GSK-3β appears to be direct, since it can be observed in vitro using purified components. Phospho-amino acid analysis indicates that the majority of Axin phosphorylation occurs on serine and threonine residues, with phosphotyrosine accounting for approximately 5% of incorporated phosphate. Interestingly, tyrosine phosphorylation of Axin by GSK-3β in kinase assays increased with increasing concentrations of ATP (data not shown). Since the cellular concentration of ATP is in order of mM, this observation suggests that tyrosine phosphorylation of Axin is more likely to be detected in vivo than in vitro. We were able to detect tyrosine phosphorylation of endogenous Axin in vivo, but only in pervanadate-treated cells, and then only at a low level (data not shown). However, pervanadate is an insulin mimetic that strongly activates PKB, which inactivates GSK-3 (35), so it is unlikely to have a strong effect on Axin tyrosine phosphorylation. Nevertheless, our results suggest that endogenous tyrosine-phosphorylated Axin is rapidly dephosphorylated by a protein- tyrosine phosphatase(s). Indeed, Axin associates with the serine/threonine phosphatase PP2A (16,53,54), and in some instances PP2A can act as a tyrosine phosphatase (55).

We have shown that FYN tyrosine kinase also phosphorylates Axin, and that the major sites of phosphorylation are the same as those phosphorylated by GSK-3β. This was somewhat surprising because the sequences surrounding Y309 and Y315 do not resemble consensus SRC family kinase phosphorylation sites. One possibility is that FYN acts through GSK-3β to elicit Axin tyrosine phosphorylation, since these proteins have been shown to interact (46). The inhibition of FYN-dependent tyrosine phosphorylation of Axin by lithium, a GSK-3β inhibitor, might be seen to support this. However, lithium also inhibits FYN-dependent phosphorylation of GFP-Axin L397P, which cannot bind to GSK-3β. Moreover, lithium has previously been reported to inhibit the effects of SRC family kinases, for example, it inhibits SRC-dependent tyrosine phosphorylation of the N-methyl-D-aspartate (NMDA) receptor NR2B subunit (56).

When we mapped the tyrosine phosphorylation sites in Axin we observed that they lie in a sequence that is related to the sequence in the activation loop of GSK-3β. The residues surrounding the tyrosine phosphorylation sites in Axin are highly conserved in the related protein, conductin/axil (4,5), and also in other vertebrate Axins, such as Xenopus Axin (57). The same region in Drosophila Axin (58,59) has no clear homology to vertebrate Axin, comprising an extended serine/threonine-rich domain, although there are candidate tyrosine phosphorylation sites nearby. The tyrosine phosphorylation sites in Axin lie about seventy residues amino-terminal to the GSK-3β-binding site (residues 373-428 (60)) and very close to S322, a previously identified GSK-3β phosphorylation site (16). The proximity of these sites to the GSK-3β-binding domain may allow them to interfere with the function of the GSK-3β autophosphorylation site (Y216) in the GSK-3β activation loop. Phosphorylation of Y216 normally causes the activation loop of GSK-3β to adopt an active conformation and defines the P1 specificity pocket for substrates (61). Without wishing to be bound by theory, the inventor considers that AxPEP may inhibit GSK-3β activity by preventing this conformational change. The physiological significance of Axin tyrosine phosphorylation remains to be . determined. Recently, Lochhead et a/ (2006) reported that GSK-3D autophosphorylates Y216 as a chaperone-dependent transitional intermediate possessing intramolecular tyrosine kinase activity. Therefore, one possibility is that Axin is involved in the maturation/folding of GSK-3 after translation; the tyrosine phosphorylation of Axin by GSK-3 during this process might then facilitate the transition of GSK-3 from an autophosphorylating tyrosine kinase into a mature serine/threonine kinase.

To conclude, we have shown that both GSK-3α and GSK-3β phosphorylate the binding partners, Axin and Axin2, on tyrosine residues. We have mapped the Axin phosphorylation sites and found that they lie in a sequence that resembles the GSK-3β autophosphorylation site. Moreover, a peptide containing the Axin tyrosine phosphorylation sites inhibits GSK-3β kinase activity, while the phosphorylated peptide does not. These observations highlight potential novel regulatory events in the Axin/GSK-3β complex, and have suggested additional peptide inhibitors of GSK-3.

References for Example 1

1. Kikuchi, A. (2000) Biochem Biophys Res Commun 268(2), 243-248

2. Polakis, P. (2000) Genes Dev 14(15), 1837-1851

3. Zeng, L., Fagotto, F., Zhang, T., Hsu, W., Vasicek, T. J., Perry, W. L., 3rd, Lee, J. J., Tilghman, S. M., Gumbiner, B. M., and Costantini, F. (1997) Ce// 90(1), 181-192

4. Behrens, J., Jerchow, B. A., Wurtele, M., Grimm, J., Asbrand, C, Wirtz, R., Kuhl, M., Wedlich, D., and Birchmeier, W. (1998) Science 280(5363), 596-599

5. Yamamoto, H., Kishida, S., Uochi, T., Ikeda, S., Koyama, S., Asashima, M., and Kikuchi, A. (1998) MoI Ce// β/o/ 18(5), 2867-2875 6. Hart, M. J., de los Santos, R., Albert, I. N., Rubinfeld, B., and Polakis, P. (1998)

CurrBiol 8(10), 573-581

7. Ikeda, S., Kishida, S., Yamamoto, H., Murai, H., Koyama, S., and Kikuchi, A.

(1998) Embo J 17(5), 1371-1384

8. Itoh, K., Krupnik, V. E., and Sokol, S. Y. (1998) CurrBiol 8(10), 591-594 9. Kishida, S., Yamamoto, H., Ikeda, S., Kishida, M., Sakamoto, I., Koyama, S., and Kikuchi, A. (1998) J Biol Chem 273(18), 10823-10826

10. Nakamura, T., Hamada, F., Ishidate, T., Anai, K., Kawahara, K., Toyoshima, K., and Akiyama, T. (1998) Genes Cells 3(6), 395-403

11. Sakanaka, C, Weiss, J. B., and Williams, L. T. (1998) Proc Natl Acad Sci U S A 95(6), 3020-3023

12. Yamamoto, H., Kishida, S., Kishida, M., Ikeda, S., Takada, S., and Kikuchi, A.

(1999) J Biol Chem 274(16), 10681-10684

13. Kishida, M., Koyama, S., Kishida, S., Matsubara, K., Nakashima, S., Higano, K., Takada, R., Takada, S., and Kikuchi, A. (1999) Oncogene 18(4), 979-985 14. Amit, S., Hatzubai, A., Birman, Y., Andersen, J. S., Ben-Shushan, E., Mann, M., Ben-Neriah, Y., and Alkalay, I. (2002) Genes Dev 16(9), 1066-1076

15. Liu, C, Li, Y., Semenov, M., Han, C, Baeg, G. H., Tan, Y., Zhang, Z., Lin, X., and He, X. (2002) Cell 108(6), 837-847

16. Yamamoto, H., Hinoi, T., Michiue, T., Fukui, A., Usui, H., Janssens, V., Van Hoof, C, Goris, J., Asashima, M., and Kikuchi, A. (2001) J Biol Chem 276(29), 26875-

26882

17. Jho, E., Lomvardas, S., and Costantini, F. (1999) Biochem Biophys Res Commun 266(1), 28-35

18. Kim, S. I., Park, C. S., Lee, M. S., Kwon, M. S., Jho, E. H., and Song, W. K. (2004) Biochem Biophys Res Commun 317(2), 478-483

19. Willert, K., Shibamoto, S., and Nusse, R. (1999) Genes Dev 13(14), 1768-1773 20. Mao, J., Wang, J., Liu, B., Pan, W., Farr, G. H., 3rd, Flynn, C₁ Yuan, H., Takada, S., Kimelman, D., Li, L., and Wu, D. (2001) MoI Cell 7(4), 801-809

21. Tolwinski, N. S., Wehrli, M., Rives, A., Erdeniz, N., DiNardo, S., and Wieschaus, E. (2003) Dev Ce// 4(3), 407-418 22. Tamai, K., Zeng, X., Liu, C₁ Zhang, X., Harada, Y., Chang, Z., and He, X. (2004) MoI Cell 13(1), 149-156

23. Zeng, X., Tamai, K., Doble, B., Li, S., Huang, H., Habas, R., Okamura, H., Woodgett, J., and He, X. (2005) Nature 438(7069), 873-877

24. Davidson, G., Wu, W., Shen, J., Bilic, J., Fenger, U., Stannek, P., Glinka, A., and Niehrs, C. (2005) Nature 438(7069), 867-872

25. Mi, K., Dolan, P. J., and Johnson, G. V. (2006) J Biol Chem 281 (8), 4787-4794

26. Swiatek, W., Kang, H., Garcia, B. A., Shabanowitz, J., Coombs, G. S., Hunt, D. F., and Virshup, D. M. (2006) J Biol Chem 281 (18), 12233-12241

27. Woodgett, J. R. (2001 ) Sci STKE 2001(100), RE12 28. Wang, Q. M., Fiol, C. J., DePaoli-Roach, A. A., and Roach, P. J. (1994) J Biol Chem 269(20), 14566-14574

29. Hughes, K., Nikolakaki, E., Plyte, S. E., Totty, N. F., and Woodgett, J. R. (1993) Embo J 12(2), 803-808

30. Kim, L., Liu, J., and Kimmel, A. R. (1999) Ce// 99(4), 399-408 31. Cole, A., Frame, S., and Cohen, P. (2004) Biochem J 377(Pt 1), 249-255

32. Plyte, S. E., Feoktistova, A., Burke, J. D., Woodgett, J. R., and Gould, K. L. (1996) MoI Cell Biol 16(1 ), 179-191

33. Malathi, K., Xiao, Y., and Mitchell, A. P. (1999) Genetics 153(3), 1145-1152

34. Zhan, X. L., Hong, Y., Zhu, T., Mitchell, A. P., Deschenes, R. J., and Guan, K. L. (2000) MoI Biol Cell 11 (2), 663-676

35. Rubin-Bejerano, I., Sagee, S., Friedman, O., Pnueli, L., and Kassir, Y. (2004) MoI Cell Biol 24(16), 6967-6979

36. Kypta, R. M., Su, H., and Reichardt, L. F. (1996) J Ce// Biol 134(6), 1519-1529

37. Sperber, B. R., Leight, S., Goedert, M., and Lee, V. M. (1995) Neurosci Lett 197(2), 149-153

38. da Costa, L. T., He, T. C, Yu, J., Sparks, A. B., Morin, P. J., Polyak, K., Laken, S., Vogelstein, B., and Kinzler, K. W. (1999) Oncogene 18(35), 5010-5014

39. Not used

40. Kodama, S., Ikeda, S., Asahara, T., Kishida, M., and Kikuchi, A. (1999) J Biol Chem 274(39), 27682-27688

41. Sehgal, R. N., Gumbiner, B. M., and Reichardt, L F. (1997) J Cell Biol 139(4), 1033-1046 42. Vivanco, M. D., Johnson, R., Galante, P. E., Hanahan, D., and Yamamoto, K. R. (1995) Embo J 14(10), 2217-2228

43. Dajani, R., Fraser, E., Roe, S. M., Young, N., Good, V., Dale, T. C, and Pearl, L. H. (2001) Ce// 105(6), 721-732 44. Kamps, M. P., and Sefton, B. M. (1989) Anal Biochem 176(1), 22-27

45. Ryves, W. J., Fryer, L., Dale, T., and Harwood, A. J. (1998) Anal Biochem 264(1), 124-127

46. Lesort, M., Greendorfer, A., Stockmeier, C₁ Johnson, G. V., and Jope, R. S. (1999) J Neural Transm 106(11-12), 1217-1222 47. Klein, P. S., and Melton, D. A. (1996) Proc Natl Acad Sci U S A 93(16), 8455- 8459

48. Stambolic, V., Ruel, L, and Woodgett, J. R. (1996) Curr Biol 6(12), 1664-1668

49. Hanke, J. H., Gardner, J. P., Dow, R. L., Changelian, P. S., Brissette, W. H., Weringer, E. J., Pollok, B. A., and Connelly, P. A. (1996) J Biol Chem 271 (2), 695-701 50. Smalley, M. J., Sara, E., Paterson, H., Naylor, S., Cook, D., Jayatilake, H., Fryer, L. G., Hutchinson, L., Fry, M. J., and Dale, T. C. (1999) Embo J 18(10), 2823-2835

51. Rubinfeld, B., Tice, D. A., and Polakis, P. (2001) J Biol Chem 276(42), 39037- 39045

52. Not used 53. Hsu, W., Zeng, L., and Costantini, F. (1999) J Biol Chem 274(6), 3439-3445

54. Ikeda, S., Kishida, M., Matsuura, Y., Usui, H., and Kikuchi, A. (2000) Oncogene 19(4), 537-545

55. Van Hoof, C, Cayla, X., Bosch, M., Merlevede, W., and Goris, J. (1994) Eur J Biochem 226(3), 899-907 56. Hashimoto, R., Fujimaki, K., Jeong, M. R., Christ, L., and Chuang, D. M. (2003) FEBS Lett 538(1 -3), 145-148

57. Hedgepeth, C. M., Deardorff, M. A., and Klein, P. S. (1999) Mech Dev 80(2), 147-151

58. Hamada, F., Tomoyasu, Y., Takatsu, Y., Nakamura, M., Nagai, S., Suzuki, A., Fujita, F., Shibuya, H., Toyoshima, K., Ueno, N., and Akiyama, T. (1999) Science

283(5408), 1739-1742

59. Willert, K., Logan, C. Y., Arora, A., Fish, M., and Nusse, R. (1999) Development 126(18), 4165-4173

60. Hinoi, T., Yamamoto, H., Kishida, M., Takada, S., Kishida, S., and Kikuchi, A. (2000) J Biol Chem 275(44), 34399-34406 61. Bax, B., Carter, P. S., Lewis, C₁ Guy, A. R., Bridges, A., Tanner, R., Pettman, G., Mannix, C, Culbert, A. A., Brown, M. J., Smith, D. G., and Reith, A. D. (2001) Structure (Camb) 9(12), 1143-1152

Additional References

Barber et al (2001) Insulin Rescues Retinal Neurons from Apoptosis by a

Phosphatidylinositol 3-Kinase/Akt-mediated Mechanism That Reduces the Activation of

Caspase-3. J Biol Chem 276:32814-821.

Bhat et al (2000). Proc Natl Acad Sci U S A, 97, 11074-9. Bijur et al (2001) Glycogen synthase kinase-3beta facilitates staurosporine- and heat shock-induced apoptosis. Protection by lithium J Biol Chem 275:7583-90 (2000).

Console et al (2003) "Antennapedia and HIV transactivator of transcription (TAT) "protein transduction domains" promote endocytosis of high molecular weight cargo upon binding to cell surface glycosaminoglycans". J. Biol. Chem. 278(37): 35109-14. Cross et al (1995). Nature, 378, 785-9.

Cross et al (2001) Selective small-molecule inhibitors of glycogen synthase kinase-3 activity protect primary neurones from death. J Neurochem 77:94-102.

Crowder et al (2000) Glycogen synthase kinase-3 beta activity is critical for neuronal death caused by inhibiting phosphatidylinositol 3-kinase or Akt but not for death caused by nerve growth factor withdrawal. J Biol Chem 275:34266-71.

De Coupade et al (2005). 2Novel human-derived cell-penetrating peptides for specific subcellular delivery of therapeutic biomolecules". Biochem. J. 390: 407-418.

Derosssi et al (1994) "The third helix of the Antennapedia homeodomain translocates through biological membranes". J. Biol. Chem. 269: 10444-50. Derosssi et al (1996) "Cell internalization of the third helix of the Antennapedia homeodomain is receptor-independent". J. Biol. Chem. 271 : 18188-93.

Deshayes et al (2005a) "Interactions of primary amphipathic cell penetrating peptides with model membranes: consequences on the mechanisms of intracellular delivery of therapeutics". Curr Pharm Des. 1 1 (28): 3629-38. Deshayes et al (2005b) "Cell-penetrating peptides: tools for intracellular delivery of therapeutics". Cell MoI Life Sci. 62(16): 1839-49.

Devlin (1997) Textbook of Biochemistry with Clinical Correlations, 4th Ed. (Wiley-Liss,

Inc)

Doble & Woodgett (2003). J Cell Sci, 116, 1175-86. Dugas et al (1981 ) Bioorganic Chemistry (Springer-Verlag, New York), pp 54-92. Eldar-Finkelman ef al (1996) "Expression and characterization of glycogen synthase kinase-3 mutants and their effect on glycogen synthase activity in intact cells", Proc Natl

Acad Sci USA 93(19):10228-10233.

Eldar-Finkelman et al (1997) "Phosphorylation of insulin receptor substrate 1 by glycogen synthase kinase 3 impairs insulin action", Proc Natl Acad Sci USA 94(18):9660-9664.

Eldar-Finkelman et al (1999) "Increased glycogen synthase kinase-3 activity in diabetes- and obesity-prone C57BL/6J mice", Diabetes 48(8): 1662-1666.

Fiol et al (1990) "Ordered multisite protein phosphorylation. Analysis of glycogen synthase kinase 3 action using model peptide substrates", J Biol Chem 265(11):6061- 6065 (1990).

Fiol et al (1994) "A secondary phosphorylation of CREB341 at Ser129 is required for the cAMP-mediated control of gene expression. A role for glycogen synthase kinase-3 in the control of gene expression", J Biol Chem 269(51 ):32187-32193 (1994).

Frame & Cohen (2001). Biochem J, 359, 1-16. Fujimuro et al (2003). Nat Med, 9, 300-6.

Grimes & Jope (2001). Prog Neurobiol, 65, 391-426.

Hanger et al (1992) Glycogen synthase kinase-3 induces Alzheimer's disease-like phosphorylation of tau: generation of paired helical filament epitopes and neuronal localisation of the kinase. Neurosci. Lett. 147:58-62. Hanks ef al (1988) Science 241 , 42-52

He et al (1995) Glycogen synthase kinase-3 and dorsoventral patterning in Xenopus embryos", Nature 374(6523):617-622. lkeda ef a/ (1998). Emho J, 17, 1371-84.

Jones ef al (2005) "Characterisation of cell-penetrating peptide-mediated peptide delivery". Br. J. Pharmacol. 145(8): 1093-1102.

Kaidanovich-Beilin & Eldar-Finkelman (2006) "Peptides targeting Protein Kinases:

Strategies and Implications". Physiology 21 : 411-418.

Klein & Melton (1996) "A Molecular Mechanism for the Effect of Lithium on

Development". Proc. Natl. Acad. Sci. USA 93:8455-8459 (1996). Lindgren et al (2000) "Cell-penetrating peptides". Trends Pharmacol. Sci. 21 (3): 99-103.

Lochhead ef al (2006) Molecular Cell 24, 627-633.

Lucas ef al (2001 ) "Decreased nuclear beta-catenin, tau hyperphosphorylation and neurodegeneration in GSK-3beta conditional transgenic mice". EMBO J 20:27-39.

Mandelkow ef al (1992) "Glycogen synthase kinase-3 and the Alzheimer-like state of microtubule-associated protein tau". Febs Lett. 314:315-21.

Mano ef a/ (2005) "On the mechanisms of the internalization of S4(13)-PV cell- penetrating peptide". Biochem J. 390(Pt 2): 603-612. Merrifield et al (1964) J Am Chem Soc 85:2149.

Morrison et al (1992) Organic Chemistry, 6th Ed. (Prentice Hall)

Mulot et al (1994) "PHF-tau from Alzheimer's brain comprises four species on SDS-

PAGE which can be mimicked by in vitro phosphorylation of human brain tau by glycogen synthase kinase-3 beta", FEBS Lett 349(3):359-364

Mulot et al (1995) "Phosphorylation of tau by glycogen synthase kinase-3 beta in vitro produces species with similar electrophoretic and immunogenic properties to PHF-tau from Alzheimer's disease brain", Biochem Soc Trans 23(1):45S.

Nonaka et al (1998) Proc. Natl. Acad. Sci. USA, 95:2642-2647. Pap & Cooper (1998) "Role of glycogen synthase kinase-3 in the phosphatidylinositol 3-

Kinase/Akt cell survival pathway". J. Biol. Chem. 273:19929-32.

Phiel (2001) "Molecular targets of lithium action". Annu Rev Pharmacol Toxicol 41 :789-

813 (2001 ).

Prochiantz (1996) "Getting hydrophilic compounds into cells: lessons from homeopeptides". Curr. Opin. Neurobiol. 6: 629-634

Rich D H (1986) in "Protease Inhibitors", Barrett and Selveson (eds) Elsevier.

Ring et a/ (2003) Diabetes 52: 588-595

Rubinfeld et al (1996) "Binding of GSK3beta to the APC-beta-catenin complex and regulation of complex assembly", Science 272(5264):1023-1026. Schiller et al (1985) lnt J Pent Prot Res 25:171.

Sherman et al (1990) J Am Chem Soc 112: 433.

Stambolic & Woodgett (1994). Biochem J, 303 ( Pt 3), 701-4.

Stambolic et al (1996) "Lithium inhibits glycogen synthase kinase-3 activity and mimics wingless signalling in intact cells". Curr. Biol. 6:1664-1668 (1996). Suzuki et al (2002) "Possible existence of common internalization mechanisms among arginine-rich peptides". J. Biol. Chem. 277(4): 2437-43.

Takeuchi et al (2006). "Direct and rapid cytosolic delivery using cell-penetrating peptides mediated by pyrenebutyrate". ACS Chem. Biol. 1 (5): 299-303.

Thomas (1995) J. Am. Geriatr. Soc, 43:1279-89. Thorsett et al (1983) "Dipeptide mimics. Conformational^ restricted inhibitors of angiotensin-converting enzyme", Biochem Biophys Res Commun 111 (1): 166-171.

Tong et al (2001 ) "Activation of glycogen syntha'se kinase 3 beta (GSK-3beta) by platelet activating factor mediates migration and cell death in cerebellar granule neurons". Eur J

Neurosci 13:1913-22. Veber et al (1978) "Conformational^ restricted bicyclic analogs of somatostatin", Proc

Natl Acad Sci USA 75(6):2636-2640. Welsh et al (1993) "Glycogen synthase kinase-3 is rapidly inactivated in response to insulin and phosphorylates eukaryotic initiation factor elF-2B", Biochem J 294(Pt 3):625- 629 (1993).

Woodgett, J.R. (2001). Sci STKE, 2001 , RE12. Yost et a/ (1998). Cell, 93, 1031-41.

Example 2: Treating a Patient with NIDDM by administering an inhibitor of GSK-3

A patient is diagnosed in the early stages of non-insulin dependent diabetes mellitus. The unphosphorylated peptide inhibitor of GSK-3, REPVNPYYVNSGYALAPATS (SEQ ID No: 5), is formulated as a cell-permeable, proteolytically resistant form in an enteric capsule. The patient is directed to take one tablet after each meal for the purpose of stimulating the insulin signaling pathway, and thereby controlling glucose metabolism to levels that obviate the need for administration of exogenous insulin.

Example 3: Treating a Patient with Alzheimer's Disease by administering an inhibitor of GSK-3

A patient is diagnosed with Alzheimer's disease. The patient is administered a pharmaceutical composition comprising the unphosphorylated peptide GSK-3 inhibitor, REPVNPYYVNSGYALAPATS (SEQ ID No: 5), which inhibits GSK-3-mediated tau hyperphosphorylation in a formulation that crosses the blood/brain barrier. The patient is monitored for tau phosphorylated polymers by periodic analysis of proteins isolated from the patient's brain cells for the presence of phosphorylated forms of tau on an SDS- PAGE gel known to characterise the presence of and progression of the disease. The dosage of the inhibitor is adjusted as necessary to reduce the presence of the phosphorylated forms of tau protein.

Example 4: Treating a Patient with Prostate Cancer by administering an inhibitor of GSK-3

A patient with prostate cancer is treated with intravenous infusions of saline solutions of a pharmaceutical composition comprising the unphosphorylated peptide GSK-3 inhibitor REPVNPYYVNSGYALAPATS (SEQ ID No: 5). The infusions are administered weekly for a time of 3 to 6 months.

Claims

1. A peptide inhibitor of glycogen synthase kinase-3 (GSK-3) comprising a peptide having between 6 and 50 amino acid residues and an amino acid sequence selected from: PYYVNSGYA (SEQ ID No: 7), EPVNPYYVNSGYALAP (SEQ ID No: 6), REPVNPYYVNSGYALAPATS (SEQ ID NO: 5), SEPVNPYHVGSGYVFAPATS (SEQ ID No: 8), EPNVSYICSRYYRAP (SEQ ID NO: 39), GEPNVSYICSTYYRAPELI (SEQ ID No: 9), EPHPSVTPTGYHAP (SEQ ID NO: 23), EPSAPS I PTAYQSS P (SEQ ID No: 24), EPPKSGDRSGYSSP (SEQ ID NO: 25), GEPPKSGDRSGYSSPGSP (SEQ ID No: 40), PFYVNSGYA (SEQ ID No: 41), EPVNPFYVNSGYA (SEQ ID No: 42), PFYVNSGYALAP (SEQ ID No: 43), EPVNPFYVNSGYALAP (SEQ ID No: 32), PYYVNSGFA (SEQ ID No: 44), EPVNPYYVNSGFA (SEQ ID NO: 45), PYYVNSGFALAP (SEQ ID No: 46), EPVNPYYVNSGFALAP (SEQ ID NO: 33), PFYVNSGFA (SEQ ID No: 47), EPVNPFYVNSGFA (SEQ ID NO: 48), PFYVNSGFALAP (SEQ ID No: 49), EPVNPFYVNSGFALAP (SEQ ID NO: 34), VNSGYA (SEQ ID No: 50), VNSGYALAP (SEQ ID No: 35), VNSGFA (SEQ ID No: 51), VNSGFALAP (SEQ ID No: 36), PYYVNSG (SEQ ID No: 52), EPVNPYYVNSG (SEQ ID No: 37), PFYVNSG (SEQ ID No: 53), EPVNPFYVNSG (SEQ ID NO: 38), EPPNYKALDFSEAP (SEQ ID No: 26), EPPVYVPPPSYRSP (SEQ ID NO: 27), EPHYYYQFTARYHAAP (SEQ ID No: 28), EPGESFCDKSYGAP (SEQ ID NO: 29), EPAPESCPPHPYPLAP (SEQ ID No: 30) or EPHPTYTDLSSHHAP (SEQ ID NO: 31) wherein the peptide is not phosphorylated at a tyrosine residue indicated by Y.

2. A peptide inhibitor of GSK-3 selected from a peptide consisting of the amino acid sequence: PYYVNSGYA (SEQ ID No: 7), EPVNPYYVNSGYALAP (SEQ ID No: 6),

REPVNPYYVNSGYALAPATS (SEQ ID No: 5), SEPVNPYHVGSGYVFAPATS (SEQ ID No: 8), EPNVSYICSRYYRAP (SEQ ID No: 39), GEPNVSYICSTYYRAPELI (SEQ ID No: 9), EPHPSVTPTGYHAP (SEQ ID No: 23), EPSAPSIPTAYQSSP (SEQ ID No: 24), EPPKSGDRSGYSSP (SEQ ID No: 25), GEPPKSGDRSGYSSPGSP (SEQ ID No: 40), PFYVNSGYA (SEQ ID No: 41), EPVNPFYVNSGYA (SEQ ID No: 42), PFYVNSGYALAP (SEQ ID No: 43), EPVNPFYVNSGYALAP (SEQ ID No: 32), PYYVNSGFA (SEQ ID No: 44), EPVNPYYVNSGFA (SEQ ID No: 45), PYYVNSGFALAP (SEQ ID No: 46), EPVNPYYVNSGFALAP (SEQ ID No: 33), PFYVNSGFA (SEQ ID No: 47), EPVNPFYVNSGFA (SEQ ID No: 48), PFYVNSGFALAP (SEQ ID No: 49), EPVNPFYVNSGFALAP (SEQ ID No: 34), VNSGYA (SEQ ID No: 50), VNSGYALAP (SEQ ID No: 35), VNSGFA (SEQ ID No: 51), VNSGFALAP (SEQ ID No: 36), PYYVNSG (SEQ ID No: 52), EPVNPYYVNSG (SEQ ID No: 37), PFYVNSG (SEQ ID No: 53), EPVNPFYVNSG (SEQ ID NO: 38), EPPNYKALDFSEAP (SEQ ID No: 26), EPPVYVPPPSYRSP (SEQ ID NO: 27), EPHYYYQFTARYHAAP (SEQ ID No: 28), EPGESFCDKSYGAP (SEQ ID NO: 29), EPAPESCPPHPYPLAP (SEQ ID No: 30) or EPHPTYTDLSSHHAP (SEQ ID NO: 31) wherein the peptide is not phosphorylated at a tyrosine residue indicated by Y.

3. A peptide inhibitor of GSK-3 comprising a peptide having between 13 and 50 amino acid residues and an amino acid sequence represented by the formula: EPX_AZ₁X_BZ₂X_C(AZS)P (SEQ ID NOS: 54-61), wherein X_A represents 2 or 3 amino acid residues which can, independently, be any amino acid residue, wherein X_B represents 4 or 5 amino acid residues which can, independently, be any amino acid residue, wherein Xc represents 1 or 2 amino acid residues which can, independently, be any amino acid residue, wherein Z₁ and Z₂, independently, represent Y, S or F, wherein (A/S) represents either A or S, and wherein the peptide is not phosphorylated at a tyrosine residue indicated by Y.

4. A peptide inhibitor of GSK-3 according to Claim 3 wherein, each independently, Z₁ is Y₁ and

Z₂ is Y.

5. A peptide inhibitor of GSK-3 according to Claim 4 wherein both Z₁ and Z₂ are Y.

6. A peptide inhibitor of GSK-3 according to any of Claims 3-5 wherein X_A consists of any 2 or all 3 residues selected from X₁X₂X₃ wherein, independently,

X₁ Js V, X₂ is N and

X₃ is P.

7. A peptide inhibitor of GSK-3 according to any of Claims 3-6 wherein XB consists of any 4 or all 5 residues selected from X₄X₅X₆X₇X₈ (SEQ ID No: 62) wherein, independently, X₄ Js Y, X₅ is V, X₆ is N, X₇ is S and X₈ is G.

8. A peptide inhibitor of GSK-3 according to any of Claims 3-6 wherein X_B represents 5 amino acid residues which can, independently, be any amino acid residue.

9. A peptide inhibitor of GSK-3 according to Claim 8 wherein X_B consists of 5 residues X₄X₅X₆X₇X₈ (SEQ ID No: 62) wherein, independently, X₄ is Y,

X₅ is V, X₆ is N, X₇ is S and X₈ is G.

10. A peptide inhibitor of GSK-3 according to any of Claims 3-9 wherein Xc consists of either 1 or 2 residues selected from X₉Xi₀ wherein, independently,

X₉ is A and X₁₀ is L.

1 1. A peptide inhibitor of GSK-3 according to any of Claims 3-10 comprising an amino acid sequence:

X₁₁ EPXAZ₁XBZ₂XC(AZS)P (SEQ ID NO: 63-70),

EPXAZ₁XBZ₂XC(AZS)PXI₂ (SEQ ID NO: 71 -78), or X₁₁ EPXAZ₁XBZ₂XC(AZS)PX₁₂ (SEQ ID NO: 79-86), wherein X₁₁ and Xi₂, independently, represent any amino acid residue.

12. A peptide inhibitor of GSK-3 according to Claim 11 wherein, independently, X₁₁ Js V and

13. A peptide inhibitor of GSK-3 according to Claim 11 or 12 comprising an amino acid sequence:

EPXAZ₁XBZ₂XC(AZS)PXI₂XI₃ (SEQ ID NO: 87-94), or X₁₁ EPXAZ₁XBZ₂XC(AZS)PX₁₂X₁₃ (SEQ ID No: 95-102), wherein X₁3 represents any amino acid residue.

14. A peptide inhibitor of GSK-3 according to Claim 13 wherein Xi₃ is T.

15. A peptide inhibitor of GSK-3 according to Claim 13 or 14 comprising an amino acid sequence: EPXAZ₁XBZ₂XC(AZS)PX₁₂X₁₃XI₄ (SEQ ID NO: 103-110), or X₁ I EPXAZ₁XBZ₂XC(AZS)PX₁₂X₁₃X₁₄ (SEQ ID NO: 111-118), wherein X₁₄ represents any amino acid residue.

16. A peptide inhibitor of GSK-3 according to Claim 15 wherein X₁₄ is S.

17. A peptide inhibitor of GSK-3 according to any of Claims 3-5 comprising the amino acid sequence:

X₁₁EPX₁X₂X₃Z₁X₄X₅X₆X₇X₈Z₂X₉X₁₀(AZS)PX₁₂X₁₃X₁₄ (SEQ ID No: 119), wherein each of X₁ to X₁₄, independently, represent any amino acid residue.

18. A peptide inhibitor of GSK-3 according to Claim 17 wherein independently, X₁ is V,

X₂ is N, X₃ is P,

X₄ is Y,

X₅ is V,

X₆ is N,

X₇ is S, X₈ is G,

X₉ is A,

X₁₀ Js L,

X₁₁ Js V,

X₁₃ is T and

X₁₄ is S.

19. A peptide inhibitor of GSK-3 according to any of Claims 1 and 3-18 further comprising the amino acid sequence of a cell-penetrating peptide.

20. A peptide inhibitor of GSK-3 according to Claim 19 wherein the cell-penetrating peptide is selected from Antennapedia_43-58; the Penetratin^™ peptide; the Antennapedia- derived, TAT-derived, Transportan-derived and polyarginine peptides listed in Table 1 of Jones et al (2005); the S4₁₃-PV and Pep-1 peptides listed in Table 1 of Mano et al (2005); the Vectocell^® penetrating peptides listed in Table 1 of de Coupade et al (2005); and the arginine-rich proteins including HIV-1 Rev and octoarginine listed in Table 1 of Suzuki et al (2005).

21. A peptide inhibitor of GSK-3 according to any of Claims 1 and 3-20 wherein the peptide is less than 30 amino acid residues in length.

22. A peptide inhibitor of GSK-3 according to any of Claims 1 and 3-20, wherein the peptide has a length of from 30 to 50 amino acid residues.

23. A peptide inhibitor of GSK-3 according to any of Claims 1-22 which has been N- terminally myristoylated, optionally via a glycine spacer.

24. A polynucleotide encoding a peptide inhibitor of GSK-3 according to any of Claims 1-22.

25. A vector comprising the polynucleotide of Claim 24.

26. A host cell comprising the polynucleotide of Claim 24 or the vector of Claim 25.

27. A recombinant cell line comprising the polynucleotide of Claim 24 or the vector of Claim 25.

28. A pharmaceutical composition comprising a peptide inhibitor of GSK-3 according to any of Claims 1-23 or the polynucleotide of Claim 24 and a pharmaceutically acceptable carrier, excipient or diluent.

29. A composition comprising a peptide inhibitor of GSK-3 according to any of Claims 1-23 or the polynucleotide of Claim 24 for use in medicine.

30. A method of combating a disease or condition mediated by GSK-3 in a patient, the method comprising administering a peptide inhibitor of GSK-3 according to any of Claims 1-23 or the polynucleotide of Claim 24, or the pharmaceutical composition of Claim 28, to the patient.

31. Use of a peptide inhibitor of GSK-3 according to any of Claims 1-23 or the polynucleotide of Claim 24 in the manufacture of a medicament for combating a disease or condition mediated by GSK-3.

32. A peptide inhibitor of GSK-3 according to any of Claims 1-23 or the polynucleotide of Claim 24 for use in combating a disease or condition mediated by GSK-3.

33. A method according to Claim 30 or a use according to Claim 31 or a peptide inhibitor/polynucleotide according to Claim 32 wherein the disease or condition mediated by GSK-3 is selected from type Il diabetes, hyperglycemia, hyperlipidemia, obesity, neurodegenerative disorders such as Alzheimer's disease, affective disorders such as bipolar disorder, manic depression and schizophrenia, neurotraumatic injuries such as acute stroke, immune potentiation, baldness or hair loss, atherosclerotic cardiovascular disease, hypertension, polycystic ovary syndrome, ischemia, brain trauma or injury, immunodeficiency, and cancer.

34. A method or a use or a peptide according to Claim 33 wherein the cancer is prostate cancer.

35. A method of inhibiting the kinase activity of GSK-3, the method comprising contacting GSK-3 with a peptide inhibitor of GSK-3 according to any of Claims 1-23.

36. A method according to Claim 33 wherein the GSK-3 is GSK-3α.

37. A method according to Claim 33 wherein the GSK-3 is GSK-3β.

38. A method according to any of Claims 35-37 that is carried out in vivo.

39. A method according to any of Claims 35-37 that is carried out in vitro.

40. A method of identifying a GSK-3 inhibitor, or a lead compound for the identification of a GSK-3 inhibitor, the method comprising: providing a candidate compound which is a peptide as defined in any of Claims 1-23; providing GSK-3, or a fragment thereof; and contacting the GSK-3 or fragment thereof with the candidate compound under conditions permitting at least one activity or function of the GSK-3 or fragment thereof; and determining whether the candidate compound inhibits the at least one activity or function of the GSK-3 or fragment thereof.

41. A method of identifying a GSK-3 inhibitor, or a lead compound for the identification of a GSK-3 inhibitor, the method comprising: providing a candidate compound; providing a polypeptide comprising or consisting of the auto-phosphorylation site of GSK-3 (EPNVSYICSTYYRAP; SEQ ID NO: 10); contacting the polypeptide with the candidate compound; and determining whether the candidate compound binds to or interacts with the polypeptide at the autophosphorylation site; wherein an agent that binds to or interacts with the autophosphorylation site of

GSK-3 is a GSK-3 inhibitor, or a lead compound for the identification of a GSK-3 inhibitor.

42. A method according to Claim 41 wherein the polypeptide comprises or consists of the amino acid sequence GEPNVSYICSTYYRAPELI (SEQ ID No: 9).

43. A method of selecting or designing a GSK-3 inhibitor, or a lead compound for the identification of a GSK-3 inhibitor, the method comprising: providing a three-dimensional structure of a peptide comprising or consisting of the amino acid sequence PYYVNSGYA (SEQ ID No: 7) that is not phosphorylated at Yj. providing a three-dimensional structure of a peptide comprising or consisting of the amino acid sequence PYYVNSGYA (SEQ ID No: 123) that is phosphorylated at Y₁ using molecular modelling means to select or design a compound that has a three-dimensional structure which is more similar to the three-dimensional structure of the unphosphorylated peptide than of the phosphorylated peptide, thereby to identify a GSK-3 inhibitor, or a lead compound for the identification of a GSK-3 inhibitor.

44. A method of selecting or designing a GSK-3 inhibitor, or a lead compound for the identification of a GSK-3 inhibitor, the method comprising: providing a comparison of the three-dimensional structure of a peptide comprising or consisting of the amino acid sequence PYYVNSGYA (SEQ ID No: 7) that is not phosphorylated at Y and of a peptide comprising or consisting of the amino acid sequence PYYVNSGYA (SEQ ID No: 123) that is phosphorylated at Yj. using molecular modelling means to select or design a compound that has a three-dimensional structure which is more similar to the three-dimensional structure of the unphosphorylated peptide than of the phosphorylated peptide, thereby to identify a GSK-3 inhibitor, or a lead compound for the identification of a GSK-3 inhibitor.

45. A method according to Claim 43 or 44 wherein the peptide comprises or consists of the amino acid sequence EPVNPYYVNSGYALAP (SEQ ID No: 6).

46. A method according to Claim 45 wherein the peptide comprises or consists of the amino acid sequence REPVNPYYVNSGYALAPATS (SEQ ID No: 5).

47. A method according to any of Claims 42-46 further comprising the step of determining whether the identified candidate compound inhibits at least one activity or function of the GSK-3.

48. A method according to any of Claims 41-47 wherein the identified compound is modified, and the modified compound is tested for inhibition of at least one activity or function of GSK-3.

49. A method according to Claims 41 , 47 or 48 wherein the at least one activity or function of the GSK-3 is kinase activity.

50. A method according to Claim 49 wherein the kinase activity of GSK-3 is tyrosine kinase activity.

51. A method according to Claim 49 wherein the kinase activity of GSK-3 is serine kinase activity.

52. A method according to any of Claims 41-51 further comprising determining whether the identified compound is selective for GSK-3α or GSK-3β.

53. A method according to any of Claims 41-52 wherein the identified compound is modified, and the modified compound is tested for selectivity for GSK-3α or GSK-3β.

54. A method according to any of Claims 41-53 wherein the identified compound is tested for inhibition of kinase activity of at least one human protein kinase other than GSK-3, preferably human CDC2.

55. A method according to any of Claims 41-54 wherein the identified compound is modified, and the modified compound is tested for inhibition of kinase activity of at least one human protein kinase other than GSK-3, preferably human CDC2.

56. A method according to Claim 54 or 55 further comprising the step of selecting a compound that has an IC₅₀ value at least 10 times lower than for the at least one other human protein kinase, preferably CDC2.

57. A method according to any of Claims 41-56 wherein the identified compound or the modified compound is tested for efficacy in a model of a disease or condition mediated by GSK-3.

58. A method according to Claim 57 wherein the model of a disease or condition mediated by GSK-3 is an animal model.

59. A method according to Claim 57 or 58 wherein the model is of a disease or condition mediated by GSK-3 as defined in Claim 33 or 34.