WO2009043116A1

WO2009043116A1 - Methods and compositions for the treatment of phosphatase- related disorders

Info

Publication number: WO2009043116A1
Application number: PCT/AU2008/001479
Authority: WO
Inventors: Helene Y. Decornez; Douglas B. Kitchen; Christopher Hovens; Grant P. Morley; Frank Guziec
Original assignee: Velacor Therapeutics Pty Ltd
Current assignee: Velacor Therapeutics Pty Ltd
Priority date: 2007-10-03
Filing date: 2008-10-03
Publication date: 2009-04-09
Anticipated expiration: 2010-04-03

Abstract

The present invention relates generally to compounds capable of modulating the activity of a protein serine/threonine phosphatase. The compounds can be used to treating conditions associated with protein serine/threonine phosphatase activity. The invention also relates to methods for screening compounds capable of modulating the activity of a protein serine/threonine phosphatase.

Description

METHODS AND COMPOSITIONS FOR THE TREATMENT OF PHOSPHATASE-

RELATED DISORDERS

FIELD OF THE INVENTION The present invention relates generally to methods and compositions for treating disorders associated with the activity of a protein phosphatase. In particular the invention includes the use of compounds based on selenium for treating conditions such as hyperproliferation (including cancer), aberrant angiogenesis, and neurological disorders. Also provided are methods for identifying compounds useful in the treatment of phosphatase-related disorders.

BACKGROUND

Protein phosphorylation (the addition of a phosphate group to a protein molecule) plays an important role in regulating many biological processes within the cell. A significant number of enzymes and receptors are activated or deactivated by phosphorylation and dephosphorylation. Phosphorylation is catalyzed by various specific protein kinases, whereas dephosphorylation is typically catalyzed by phosphatases. Thus, a phosphatase is an enzyme that removes a phosphate group from its substrate by hydrolysing phosphoric acid monoesters into a phosphate ion and a molecule with a free hydroxyl group (see dephosphorylation). This action is directly opposite to that of phosphorylases and kinases, which attach phosphate groups to their substrates by using energetic molecules like ATP.

An example of the important interplay between kinases and phosphatases is the p53 tumour suppressor gene which, when active, stimulates transcription of genes that suppress the cell cycle, even to the extent that the cell undergoes apoptosis.

However, this activity must be carefully controlled and limited to situations where the cell is damaged or its physiology is disturbed. To this end, the p53 protein is extensively regulated. In fact, p53 contains more than 18 different phosphorylation sites. Upon the deactivating signal, the protein is dephosphorylated again and ceases operation. It is widely accepted that disregulated protein phosphorylation is a frequent cause of disease, particularly cancer, where kinases and phosphatases regulate many aspects that control cell growth, movement and death. In consideration of that fact, there have been many previous attempts at identifying compounds capable of binding to and altering the biological activity of clinically relevant protein phosphatases. The present invention seeks to provide alternative compounds to those already identified, and also alternative medical uses for known compounds based on the ability of those compounds (whether known or not ) to regulate the activity of a protein phosphatase. In the context of use as a therapeutic, the compounds may demonstrate improvements over the prior art in the areas of efficacy, side effect profile, applicability, pharmacodynamics, ease of administration and the like.

The present invention further seeks to provide binding motifs on protein serine/threonine phosphatases that are capable of binding ligand to alter the activity of the enzyme. Such binding motifs are useful for screening for compounds capable of modulating protein serine/threonine phosphatase activity. It is an aim of the present invention to identify such binding sites for use in screening compounds with the potential to modulate the activity of protein serine/threonine phosphatases such as PP2A. Such compounds could increase or decrease the level of dephosphorlyation of certain proteins.

There is a further need in the art to identify agents that are capable of greater in treating or preventing disorders related to protein serine/threonine phosphatase activity. Such agents may provide for a lessened side effect profile, as distinct from other agents having indiscriminate or pleiotropic effects.

The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

SUMMARY OF THE INVENTION

The present invention provides compounds useful in the treatment of a range of conditions associated with protein phosphatase activity. In a first aspect, the present invention provides a compound capable of modulating protein serine/threonine phosphatase activity, the compound having the formula X(R¹)m(R²)n(R³)p(R⁴)_q or

wherein X is selected from the group consisting of sulphur, selenium, and tellurium;

R¹ R², R³ and R⁴ are each independently selected from the group consisting of: =0, =S, OR⁵, SR⁵, NR⁵, H, optionally substituted C₁-C₁₂ alkyl, optionally substituted C₂-C₁₂ alkenyl, optionally substituted C₂-C₁₂ alkynyl, optionally substituted C₁-C₁₀ heteroalkyl, optionally substituted C₃-C₁₂ cycloalkyl, optionally substituted C₃-C₁₂ cycloalkenyl, optionally substituted C₂-

C₁₂ heterocycloalkyl, optionally substituted C₂-C₁₂ heterocycloalkenyl, optionally substituted C₆-C₁₈ aryl, optionally substituted CrC₁₈ heteroaryl, and acyl;

each R⁵ is independently selected from the group consisting of: H, optionally substituted CrC₁₂ alkyl, optionally substituted C₂-C₁₂ alkenyl, optionally substituted C₂-C₁₂ alkynyl, optionally substituted CrC₁₀ heteroalkyl, optionally substituted C₃-C₁₂ cycloalkyl, optionally substituted C₃-C₁₂ cycloalkenyl, optionally substituted C₂-C₁₂ heterocycloalkyl, optionally substituted C₂-C₁₂ heterocycloalkenyl, optionally substituted C₆-C₁₈ aryl, optionally substituted CrC₁₈ heteroaryl, and acyl;

m is an integer selected from 0 and 1 ,

n is an integer selected from 0 and 1 ,

p is an integer selected from 0 and 1 ,

q is an integer selected from 0 and 1 , the sum of m+n+p+q is an integer from 2 to 4,

or a pharmaceutically acceptable salt thereof.

In one embodiment of the compound, X is selenium.

In one embodiment of the compound, n is 1. In another embodiment n is 2.

In one embodiment of the compound, the compound is selenium dioxide. The selenium dioxide may have the following structure:

In one embodiment of the compound, the compound is selected from the group consisting of

or a pharmaceutically acceptable salt thereof.

In another embodiment of the compound, the compound is selected from the group consisting of

or a pharmaceutically acceptable salt thereof.

In one embodiment of the compound, R¹ is selected from the group consisting of H, CH₃, CH₂CH₃, CH₂CH₂CH₃, CH(CH₃)₂, CH₂CH₂CH₂CH₃, C(CH₃)₃,and phenyl.

In one embodiment of the compound, R² is selected from the group consisting of H, CH₃, CH₂CH₃, CH₂CH₂CH₃, CH(CH₃)₂, CH₂CH₂CH₂CH₃, C(CH₃)₃,and phenyl.

In one embodiment of the compound, the compound is selected from the group ccoonnssiissttiinngg ooff SSOO₄₄ ^22"",, SSeeOO₄₄ ^22"",, SSeeOO₂₂((CCHH₃)₂, SeO₃CH₃ ^", SeO₂CH₃ ^", SeO₃SH^", or a pharmaceutically acceptable salt thereof.

Without wishing to be limited by theory in any way, a compound as described herein may be useful in the treatment of certain diseases by an ability to modulate (and particularly upregulate) the activity of a protein serine/threonine protein phosphatase such as PP1 α, PP1 β, PP1γ1 , PP1γ2, PP2A, PP2B (also known as calcineurin) , PP2C, PP4, PP5 and PP6. In particular, the compound may be capable of upregulating PP2A.

In another aspect, the present invention provides a composition comprising a compound as described herein in combination with a pharmaceutically acceptable carrier.

In a further aspect the present invention provides a method for upregulating the activity of a serine/threonine protein phosphatase, the method comprising the step of exposing the cell to an effective amount of a compound as described herein, or a composition as described herein. The protein serine/threonine phosphatase may be PP1 α, PP1 β, PP1γ1 , PP1γ2, PP2A, PP2B, PP2C, PP4, PP5 or PP6. In one embodiment of the invention the protein serine/threonine phosphatase is PP2A.

In a further aspect, the present invention provides a method for treating or preventing a condition associated with the activity of a serine/threonine protein phosphatase, the method comprising administering to a subject in need thereof a therapeutically effective amount of a composition as described herein. The protein serine/threonine phosphatase may be PP1 α, PP1 β, PP1γ1 , PP1γ2, PP2A, PP2B, PP2C, PP4, PP5 or PP6. In one embodiment of the invention the protein serine/threonine phosphatase is PP2A.

The condition associated with the activity of a serine/threonine protein phosphatase may be a hyperproliferative disorder (such as cancer), a vascular hyperproliferative disorder (such as atherosclerosis), or a neurological disorder (such as Alzheimer's disease).

The condition associated with the activity of a serine/threonine protein phosphatase may also be a non-tumor disease or disorder associated with aberrant angiogenesis, such as macular degeneration.

In another aspect, the present invention provides use of a compound as described herein in the manufacture of a medicament for the treatment or prevention of a condition associated with the activity of a serine/threonine protein phosphatase. In a further aspect the present invention provides a method of identifying a compound capable of modulating protein serine/threonine phosphatase activity, the method comprising:(a) contacting the protein serine/threonine phosphatase or an analogue or fragment thereof with a candidate compound under conditions permitting binding of the test compound to the protein serine/threonine phosphatase; and (b) determining whether the test compound binds to a binding motif on the catalytic subunit of the protein serine/threonine phosphatase or to an analogue or fragment thereof. In one embodiment the method comprises determining whether the candidate compound modulates protein serine/threonine phosphatase activity. The protein serine/threonine phosphatase may be PP1 α, PP1 β, PP1γ1 , PP1γ2, PP2A, PP2B, PP2C, PP4, PP5 or PP6, but is particularly PP2A.

In certain embodiments of the method the binding motif comprises one or more amino acids from any one of the amino acid clusters selected from the group consisting of:

(a) D57, H59, D85, R89, N117, H118, H167, R214, H241;

(b) D57, H59, D85, N117, H167, H241;

(c) 162,163,235,236,250,254,278,282,284; (d) N1 17,H1 18,S120,R121 ,Q122,l 123,T124,E188,V189,P190,H191 ,G192,C196, W200,A216, G217;

(e) I 14,N18,M66,F69,R7O,G73,K74,S75,Y8O,L99,L1 O3;

(f) F164,L166,G169,L17O,S171 ,I 174,I 18O,L198,W2O9,F228;

(g) L39,Y86,V97,V101 ,l1 13,E1 19,F146,L149,F150,L153; (h) Y86,T96,V97,L100,V101 ,l 1 13,F150,L153;

(i) Q46,E47,V48,R49,P51 ,V52,N79,Y80,L81 ,K104,R108,E109,R1 10,11 1 1.T1 12;

C) L31 ,A35,L39,L100,V101 ,K104,V105,l1 13,L149,L153;

(k) D57,V58,Q61 ,D64,L65,L68,S261 ,A262,P263,F289;

(I) C55,G56,D57,V58,H59,L81 ,F82,M83,G84,D85,Y86,L100,L1 14,S261 ; (m) D57,H59,D85,R89,N1 17,H1 18,l123,Y127,W200,R214,H241 ,F260,Y265;

(n) V28,K29,C32,E33,K36,K144,Y145,D148,L149;

(o) I 14,S75,P76,L1 O3,R1 O6,Y1 O7,I1 1 1 ;

(P) L39,T40,D151 ,Y152,L153,R185,L186,Q187;

(q) H191 ,E192,G193,C196,D197,A216,G217,Y218; (r) Y107,R108,E109,R1 10;

(s) P51 ,V52,T53,D77,T78,N79,Y80,M276,E277,L278,D279 ; (t) D131 ,L134,R135,Y307,F308;

(u) V244,M245,E246,G247,Y248,N249,W250;

(v) W200,S201 ,D202,S212,P213,R214,A216,G217,Y218,T219,F220;

(w) R89,R214,H241 ,Q242,L243,F260,Y265; (X) F6,W13,S30,L31 ,E33,K34,E37;

(y) L10,D1 1 ,l14,E15,P76,R106,Y107;

(Z) G90,Y91 ,Y92,S93,G128,F129,D131 ,E132,R135,K136;

(aa) L17O,S171 ,I174,D175,W2O9,I224,T227,F228;

(bb) H59,D85,D88,R89,H1 18,Y127,Y265; (CC) Y92,Y267,K294,R295,G296,E297,P298,V308;

(dd) P203,D204,D205,R206,G207,G221 ,Q222,D223,Q242,N249,C251 ,H252,D253;

(ee) L183,D184,R185,Q187,P190,P194,H195;

(ff) Y267,P291 ,A292,R294;

(gg) P203, R239,Q242, L243,V244, N249.T258; (hh) E67,l71 ,F289,D290,P291 ,A292,P293;

(ii) E33,K36,E37;

Cj) V5,F6,T7,K8,E9;

(kk) I21 1 ,S212,G215,A216,G217,Y218;

(II) E226,N229,H230,L234,N255; (mm) T176,L177,D178,R181 ,N232;

(nn) H63,H66,E67,R70,A292,P293,R294;

(oo) H63,Y92,Y267,R294,R295;

(PP) D202,P203,D204,P213.T219,F220,Q242;

(qq) V300,R303,T304,P305; (rr) H63,P293,R294,R295;

(SS) T40,N44,L183,D184,R185,L186;

(tt) E42,Q46,V48,K104,R108,E109,l111,T112;

(UU) D204,R206,G210,l21 1 ,S212,P213,T219;

(W) D204,R206,l211 ,P213,T219; and (WW) N18,E19,C20,F62,H62,M63

(XX) R89, N117, H1 18, 1123, Y127, W200, P213, R214, H241 , Q242, L243, F260,

Y265;

(yy) N117, S120, R121, Q122, 1123, Y127, E188, V189, P190, H191, W200;

(ZZ) D204, D205, C221, Q222, G251, H252, D253; (aaa) R89, R214;

(bbb) H191.Q122; (CCC) E188, R121 ; (ddd) H252, D253; and (eee) D290.

In one form of the method, the binding motif is a component of a binding complex, the binding complex comprising an accessory atom, ion or molecule. In a further embodiment the accessory ion is Mn2+.

In one embodiment, the method comprises the step of isolating the test compound that has been identified as capable of modulating protein serine/threonine phosphatase activity.

In a further aspect the present invention provides a method of identifying a compound which is capable of binding to a protein serine/threonine phosphatase, the method comprising: (a) providing a three dimensional structure of a catalytic subunit of a protein serine/threonine phosphatase, or an analogue or fragment thereof; and (b) identifying a candidate compound for binding to one or more binding motifs on the catalytic subunit, or the analogue or fragment thereof by performing structure-based drug design with the structure of (a).

Yet a further aspect of the invention provides a method of identifying a candidate compound capable of binding to a protein serine/threonine phosphatase, the method comprising: (a) identifying a candidate compound that has a conformation and polarity such that it interacts with at least one relevant amino acid residue of one or more binding motifs on a catalytic subunit of the protein serine/threonine phosphatase, or an analogue or fragment thereof. It will be understood that this aspect includes the situation whereby a PP2A is exposed to a candidate compound in the context of a cell, cell lysate, at least partially purified form of PP2A, or recombinantly purified form of PP2A.

A further aspect of the invention provides a computer-assisted method for identifying a candidate compound capable of binding to a protein serine/threonine phosphatase, the method comprising: (a) supplying a computer modelling application with a set of structure coordinates of a molecule or molecular complex, at least a portion of the structural coordinates of the molecule or molecular complex being derived from, or defining the same relative spatial configuration as, at least a portion of the atomic coordinates of one or more binding motifs on a catalytic subunit of the protein serine/threonine phosphatase; (b) supplying the computer modelling application with a set of structure coordinates of the candidate compound; and (c) determining whether the candidate compound is expected to bind to the molecule or molecular complex, wherein binding to the molecule or molecular complex is indicative of potential binding to the catalytic subunit of the protein serine/threonine phosphatase.

In another aspect, the present invention provides a computer-assisted method for designing a candidate compound capable of binding to a catalytic subunit of a protein serine/threonine phosphatase, the method comprising: (a) supplying a computer modelling application with a set of structure coordinates of a molecule or molecular complex, at least a portion of the structural coordinates of the molecule or molecular complex being derived from, or defining the same relative spatial configuration as, at least a portion of the atomic coordinates of one or more of the binding motifs of the catalytic subunit of the protein serine/threonine phosphatase; (b) supplying the computer modelling application with a set of structure coordinates for the candidate compound; (c) evaluating the potential binding interactions between the candidate compound and substrate binding pocket of the molecule or molecular complex; (d) structurally modifying the candidate compound to yield a set of structure coordinates for a modified candidate compound; and (e) determining whether the modified candidate compound is expected to bind to the molecule or molecular complex, wherein binding to the molecule or molecular complex is indicative of potential binding to the catalytic subunit of the protein serine/threonine phosphatase.

In another aspect the present invention provides a computer-assisted method for designing a candidate compound capable of binding to a catalytic subunit of a protein serine/threonine phosphatase de novo, the method comprising: (a) supplying a computer modelling application with a set of structure coordinates of a molecule or molecular complex, at least a portion of the structural coordinates of the molecule or molecular complex being derived from, or defining the same relative spatial configuration as, at least a portion of the atomic coordinates of one or more of the binding motifs of the catalytic subunit of the protein serine/threonine phosphatase; (b) computationally building a candidate compound represented by a set of structure coordinates; and (c) determining whether the candidate compound is expected to bind to the molecule or molecular complex, wherein binding to the molecule or molecular complex is indicative of potential binding to the catalytic subunit of the protein serine/threonine phosphatase.

In one embodiment of the computer-assisted method, the three dimensional structure of the catalytic subunit of the protein serine/threonine phosphatase is provided by the atomic coordinates provided in the prior art :An exemplary crystal structure and coordinates are disclosed in Cho and Xu, "Crystal structure of a protein phosphatase 2A heterotrimeric holoenzyme" Nature 445:53-57, 2007 and as publicly disclosed in the RCSB Protein Databank under accession 2iae

(http://www.rcsb.org/pdb/files/2iae.pdb); as accessed 7 December 2007; the contents of both being herein incorporated by reference.

The step of identifying a candidate compound for binding to one or more binding motifs on the catalytic subunit may be performed using a fitting operation between the three-dimensional structure of the one or more binding motifs and the candidate compound. In a particular form of the method, the candidate compound demonstrates a docking score of at least about -2.5, and preferably less than about -7.5.

The one or more binding motifs may be any of those listed supra from (a) to (eee).

Further, the method may comprise the step of obtaining the candidate compound, contacting the candidate compound with a protein serine/threonine phosphatase, or an analogue, a homolog or a fragment thereof and determining the ability of the candidate compound to modulate protein serine/threonine phosphatase activity. The step of determining the ability of the candidate compound to modulate protein serine/threonine phosphatase activity may comprise determining the ability of the candidate compound to enhance protein serine/threonine phosphatase activity. Alternatively, the step of determining the ability of the candidate compound to modulate protein serine/threonine phosphatase activity comprises determining the ability of the candidate compound to inhibit protein serine/threonine phosphatase activity.

In a further aspect the present invention provides a method of screening to identify a compound capable of modulating protein serine/threonine phosphatase activity comprising: contacting a sample containing a PP2AB subunit complex of the protein serine/threonine phosphatase with a compound to be screened; and determining a differential effect of the compound on a PP2AB form of PP2A in the presence and absence of the compound.

In one embodiment of the method the compound is capable of selectively regulating the phosphatase activity of the PP2AB form of PP2A.

In another embodiment of the method, prior to determining the differential effect of the compound on a PP2AB form of PP2A in the presence and absence of the compound, a differential effect of the compound on phosphatase activity of PP2A in general is determined for comparison against phosphatase activity of the PP2AB form. The differential effect may be measured as an increase or decrease in phosphatase activity of the PP2AB compared to a control in the absence of the compound. Furthermore, the compound to be identified may regulate the phosphatase activity by activating the PP2A and/or PP2AB activity.

In one form of the method the differential effect may be determined by a measure of dephosphorylation of a phosphopeptide/protein, and particularly protein kinase Akt. Alternatively, the differential effect may be measured by dephosphorylation of tau protein.

In certain embodiments of the method, the PP2AB form of PP2A is PP2ABα, PP2ABβ, PP2ABγ or PP2AB5. The sample used in the method may be a cell, a cell lysate, a nucleic acid, a protein or a peptide.

Also provided by the present invention is a compound identified by the above methods, and also a composition comprising such a compound in combination with a pharmaceutically acceptable carrier. Another form of the invention provides a method of treating a condition associated with protein serine/threonine phosphatase activity in a patient in need, said method comprising administering an effective amount of such a composition

In another aspect the present invention provides a method of treating a condition associated with protein serine/threonine phosphatase activity in a patient in need, said method comprising: identifying a compound that selectively regulates phosphatase activity of a form of PP2A, said method comprising: contacting a sample containing PP2AB subunit complex with a compound to be screened; and determining a differential effect of the compound on a PP2AB form of PP2A in the presence and absence of the compound; and administering an effective amount of the identified compound to the patient in need.

The condition associated with protein serine/threonine phosphatase activity is a hyperproliferative disorder, such as cancer. The cancer may be a cancer of the brain, lung, liver, spleen, kidney, lymph node, small intestine, pancreas, blood cells, colon, stomach, breast, endometrium, prostate, testicle, ovary, skin, head and neck, esophagus, bone marrow and blood cancer; carcinomas (comprising colon, prostate, breast, melanoma, ductal, endometrial, stomach, dysplastic oral mucosa, invasive oral cancer, non-small cell lung carcinoma, transitional and squamous cell, urinary carcinoma); neurological malignancies (comprising neuroblastoma, gliomas).; hematological malignancies, (comprising childhood acute leukaemia, non-Hodgkin's lymphomas, chronic lymphocytic leukaemia, malignant cutaneous T-cells, mycosis fungoides, non-MF cutaneous T-cell lymphoma, lymphomatoid papulosis, T-cell rich cutaneous lymphoid hyperplasia, bullous pemphigoid, discoid lupus erythematosus, lichen planus; sarcomas, melanomas, adenomas; benign lesions such as papillomas, and the like, uterine, testicular and ovarian carcinomas, endometriosis, squamous or glandular epithelial carcinomas of the cervix.

In another form of the method of treatment the hyperproliferative disorder is a vascular hyperproliferative disorder, and in particular restenosis , in-stent stenosis, and vascular graft restenosis, neointimal occlusive lesions, atherosclerosis, graft coronary vascular disease after transplantation, vein graft stenosis, peri-anastomatic prosthetic graft stenosis, restenosis after angioplasty or stent placement.

In other embodiments of the method the hyperproliferative disorder is a fibrotic disorder, an inflammatory disorder (comprising arthritis), endometriosis; psoriasis, a benign growth disorder (comprising prostate enlargement and lipoma); or an autoimmune disorder (comprising scleroderma, systemic lupus erythematosus, Sjogren's syndrome, atopic dermatitis, asthma, and allergy).

In one form of the invention the condition associated with protein serine/threonine phosphatase activity is a neurodegenerative condition, and in particular diseases such as Creutzfeldt Jakob disease, Huntington's disease, stroke, cerebral ischaemia, dementia associated with stroke or cerebral ischaemia, dementia associated with HIV, disorders associated with excitotoxicity, epilepsy, seizures, schizophrenia, bipolar disorder, depression, mood disorder, multiple sclerosis, acute brain trauma (severe traumatic brain injury) or motor neurone disease.

In another embodiment of the method the neurodegenerative condition is presenile dementia, senile dementia, Alzheimer's disease, Parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy (PSP), Pick's disease, primary progressive aphasia, frontotemporal dementia, corticobasal dementia, Parkinson's disease, dementia with Lewy bodies, Down's syndrome, multiple system atrophy, amyotrophic lateral sclerosis (ALS), multiple sclerosis, or Hallervorden Spatz syndrome.

The condition associated with protein serine/threonine phosphatase activity may be a non-tumor disease or disorder associated with aberrant angiogenesis, for example a clinical condition characterised by excessive vascular endothelial cell proliferation, vascular permeability, edema or inflammation comprising macular degeneration, especially age-related macular degeneration comprising wet macular degeneration and dry macular degeneration; a retinopathy such as diabetic retinopathy, ischaemic retinal vein occlusion and retinopathy of prematurity; endometriosis, restenosis, psoriasis and rheumatoid arthritis, brain edema associated with injury, stroke or tumor, edema associated with inflammatory conditions such as psoriasis, and arthritis comprising rheumatoid arthritis, asthma, generalised edema associated with burns, ascites and pleural effusion, macular degeneration, diabetic retinopathy, endometriosis or restenosis.

Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the human PP2A holoenzyme crystal structure reported by Cho and Xun (Nature 445:53-57; 2007). The crystal structure shows a complex of an A (scaffold) subunit, a B' (regulatory) subunit and a C (catalytic) subunit. The PP2A inhibitor microcystin is also shown. The A subunit contains 15 HEAT repeats, forming a horseshoe shape. There is a substantial conformational change from the twisted hook shape of the monomeric structure to a more closed C-shape of the trimeric structure of the A subunit. The B' subunit also contains eight pseudo HEAT repeats. The C subunit forms a compact ellipsoidal structure and contains 2 Mn2+ ions.

Figure 2 is a graphical representation of the crystal structure of sulfate (A) and selenate (B) complexed on the catalytic subunit of PP2A using a docking score cut-off of -2.5, and preferably less than about -7.5.

Figure 3 is a graphical representation of a number of compounds modelled on the binding motifs on the catalytic subunit of the protein serine/threonine phosphatase, PP2A.

Figure 4 shows a graphical overlay of a PP2A active site (grey; pdbcode: 2IE3) against PP5 (blue; pdbcode: 1S95). The active active site residues are conserved (grey numbering PP2A; blue numbering PP5). PP5 structure shows a phosphate directly ligated to the two Mn²⁺ ions. Similar positioning of a selenate group in the PP2A active site is a second attractive location.

Figure 5 is a graphical representation of two hypotheses for selenate binding to one of the binding motifs on the catalytic subunit of PP2A (A and B). Though the two positions are close, the difference arises from whether the selenate interacts directly with the Mn²⁺ ions or whether that interaction is mediated or shielded by a water molecule. These water molecules are frequently seen in the various PP2A, PP1 and PP5 structures. However, not all waters are seen in the crystal structures. There are usually six species ligated to the metal centers, and for binding site one, two waters are present at the spot occupied by phosphate oxygens in the PP5 structure. This PP5 structure is the basis for Hypothesis 2 which simply puts a sulfate/selenate in place of the phosphate. In some enzymes, Mn and Mg can play almost identical roles, while not in others (see, for example, Bock et. al., J. Am. Chem. Soc. 1999, 121 :7360-7372). These ions are likely to play a role in the catalytic activity of the PP2A site and their locations appear to be stabilized by several aspartic acids and histidines. Hypothesis based on binding of inhibitors with overlapped acidic groups. In Figure 5A, selenate is positioned as an acidic group replacement and stabilized by tyrosine 262 and arginine 214. Selenate and additional water molecule (italic) can compete to interact with the Mn2+ ions. The Hypothesis depicted in Figure 5B is derived from the interaction of the phosphate group with divalent Mn²⁺ in PP5. Selenate is shown here in direct contact with Mn²⁺. The interactions between the metal ions and its ligands are indicated by dashed lines. The amino acids in the binding motif are numbered as in PP2A structure from PDB code 2IAE.

Figure 6 shows a graphical representation of the structure of a selenate ion used for the parameterization of the force field (charges are indicated in red, distances are indicated in grey). Quantum mechanical (QM) optimization of a selenate with a

LAV2P^* basis set was used to obtain the atomic charges from Mulliken population and bond distances. Parameters were incorporated for QM/MM calculations with QSite

(Schrodinger, LLC, New York, NY, Glide v4.5 .108) and for molecular mechanics (MM) calculations with Impact (Schrodinger, LLC, New York, NY, jaguar v1.165.2.2) and Glide (Schrodinger, LLC, New York, NY, qsite v1.6.4.1 ) for molecular docking.

Figure 7 shows the validation of selenate parameterization. A comparison of small systems shows minimal changes in bond distances (grey) and charges (red). All QM optimizations use the LAV2p^* basis with DFT B3LYP. All QM/MM calculations use the LAV2p^* basis with DFT B3LYP for the QM region and the modified OPLSAA2001 force field for the MM region. All MM calculations use the modified OPLSAA2001 force field.

Figure 8 is a graphical representation of the protein configuration of PP2A showing the binding energy contributions of sulfate and selenate to the PP2A catalytic chain. The figure on the left is a two dimensional graphic representation of the active site interactions. The figure on the right is a three dimensional graphic representation of the active site of the original crystal structure (grey carbon atoms) and minimized structure (green carbon atoms; waters omitted for clarity). Prepared protein shows few variations from the initial crystal structure. Selenate is shown here docked between amino acid residues R89 and R214. Mg2+ ions are depicted here as purple spheres.

Figure 9 represents the QM/MM approach in illustrating the binding energy contributions of sulfate and selenate to the PP2A catalytic chain. Docked sulfate and selenate with Glide was performed to obtain initial position in the prepared protein active site (binding motif). Shells of waters were then added around the sulfate/selenate. We then minimized the structure with Impact for input to QM/MM optimization. QM/MM structure optimization: (i) QM region: sulfate/selenate, free to move; (ii) MM region: protein and water, free to move: waters, R89 and R 214, Y167,

Y265. The structure depicted in this Figure is the resulting QM/MM optimized structures: sulfate structure (yellow sulfur), carbon atoms (grey), water molecules (red), selenate structure (selenium: blue), carbon atoms (green) and water molecules (maroon).

Figure 10 is a graphical representation of the potential sulfate binding sites on PP2A catalytic subunit illustrating the location of the binding pockets (motifs) on the surface of the catalytic subunit of PP2A (pdbcode 2IE4). Binding sites were identified using SiteFinder in MOE1. There were 47 sites identified as having the shape to bind small ions such as sulfate or selenate. The catalytic subunit is shown here colored by electrostatic potential. The binding pockets (motifs) are identified by clusters of dummy atoms (dark grey). The catalytic active site is identified by Mn²⁺ ions (green spheres). All sites were prepared for docking studies. Grids were built for each catalytic active site and residues within 1θA from the scaffolding and regulatory subunits of PP2A to prevent placement of sulfate or selenate in region crucial for PP2A function. Selenate and sulfate were docked with Glide^* XP and 20 top scoring poses for each ligand investigated per site.

Figure 11 shows the potential sulfate and selenate binding motifs on the catalytic subunit of PP2A. The location of sulfates (A) and selenates (B) on the catalytic site of PP2A resulting from docking studies to the 47 sites previously identified are shown. Most sulfates and selenates bind in similar locations. The main site investigated is still favorable for binding of a sulfate/selenate, but other sites can be further considered. Using a cutoff of -7.5 on the docking scores and ignoring sites with a neutral aspartic acid or glutamic acid, the following motifs are also of interest (see Tables 3-5): 2,5,16,28,32,40 (sites 9,10,11 ,21 ,26 correspond to different regions of the main site investigated).

Figure 12 is a graphical representation of the structure and charges of selenones. Charges and geometries of selenones were calculated as previously for selenate. The selenone systems have smaller partial charges with less polarized bonds as compared to selenate. The selenones are thus expected to be good mimics of selenate.

Figure13 shows enhancement of phosphatase activity of PP2A using inorganic selenium species. A) 0.01 U of PP2A AC core dimmer was incubated with 500 μM phospho-threonine peptide with the indicated selenium species (all at 50 μM) for 15 min at 37°C. Free phosphate was assayed using the malachite green assay. Absorbance readings at 590nm were recorded and plotted. B) 0.01 U of PP2A AC core dimmer was incubated with 500 μM phospho-threonine peptide with the indicated selenium species (all at 50 μM) for 15 min at 37°C. Free phosphate was assayed using the malachite green assay. Relative phosphatase activities of the various treatments are plotted.

Figure 14A shows location of sites 2 and 10 on PP2A from pdbcode 21 E4. Okadaic acid is shown in yellow and the electrostatic potential surface is shown around PP2A. 14B shows a rotated view of showing location of sites 28 and 32 as well as site 10 for reference (pdbcode 21 E4). Okadaic acid is shown in yellow and the electrostatic potential surface is shown around d PP2A.

Figure 15 shows three alternate docked location of selenate in site 10 of optimized PP2A (pdbcode 2IE4).

Figure 16 shows location of selenate in site 2/15 of optimized PP2A (pdbcode 2IE4).

Figure 17 shows docked location of selenate in site 28/40 of optimized PP2A (pdbcode 2IE4).

Figure 18 shows docked location of selenate in site 32 of optimized PP2A (pdbcode 2IE4).

Figure 19 shows parameterization of selenate and selenite.

Figure 20 shows parameterization of selenium dixoide.

DETAILED DESCRIPTION OF THE INVENTION The present invention is predicated in part of the inventors' discovery that the activity of a serine/threonine protein phosphatase can be increased by binding of a compound to a newly identified binding motif on the surface of a catalytic subunit of a protein serine/threonine phosphatase. The compound may be based on sulphur, selenium, or tellurium. Accordingly, in a first aspect the present invention provides a compound capable of modulating protein serine/threonine phosphatase activity, the compound having the formula: X(R¹)m(R²)n(R³)p(R⁴)q

or

R¹ R², R³ and R⁴ are each independently selected from the group consisting of: =0, =S, OR⁵, SR⁵, NR⁵, H, optionally substituted C₁-C₁₂ alkyl, optionally substituted C₂-C₁₂ alkenyl, optionally substituted C₂-C₁₂ alkynyl, optionally substituted C₁-C₁₀ heteroalkyl, optionally substituted C₃-C₁₂ cycloalkyl, optionally substituted C₃-C₁₂ cycloalkenyl, optionally substituted C₂- C₁₂ heterocycloalkyl, optionally substituted C₂-C₁₂ heterocycloalkenyl, optionally substituted C₆-C₁₈ aryl, optionally substituted C₁-C₁₈ heteroaryl, and acyl;

each R⁵ is independently selected from the group consisting of: H, optionally substituted C₁-C₁₂ alkyl, optionally substituted C₂-C₁₂ alkenyl, optionally substituted C₂-C₁₂ alkynyl, optionally substituted C₁-C₁₀ heteroalkyl, optionally substituted C₃-C₁₂ cycloalkyl, optionally substituted C₃-C₁₂ cycloalkenyl, optionally substituted C₂-C₁₂ heterocycloalkyl, optionally substituted C₂-C₁₂ heterocycloalkenyl, optionally substituted C₆-C₁₈ aryl, optionally substituted C₁-C₁₈ heteroaryl, and acyl;

m is an integer selected from 0 and 1 ,

n is an integer selected from 0 and 1 ,

p is an integer selected from 0 and 1 , q is an integer selected from 0 and 1 ,

the sum of m+n+p+q is an integer from 2 to 4,

or a pharmaceutically acceptable salt thereof.

In one embodiment of the compound, X is selenium.

In one embodiment of the compound, n is 1. In another embodiment n is 2.

In one embodiment of the compound, the compound is selenium dioxide (SeO₂).

In one embodiment of the compound, the compound is selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

or a pharmaceutically acceptable salt thereof.

In this specification a number of terms are used which are well known to a skilled addressee. Nevertheless for the purposes of clarity a number of terms will be defined. As used herein, the term unsubstituted means that there is no substituent or that the only substituents are hydrogen.

The term "optionally substituted" as used throughout the specification denotes that the group may or may not be further substituted or fused (so as to form a condensed polycyclic system), with one or more non-hydrogen substituent groups. In certain embodiments the substituent groups are one or more groups independently selected from the group consisting of halogen, =0, =S, -CN, -NO₂, -CF₃, -OCF₃, alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl, haloalkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, heteroarylalkyl, arylalkyl, cycloalkylalkenyl, heterocycloalkylalkenyl, arylalkenyl, heteroarylalkenyl, cycloalkylheteroalkyl, heterocycloalkylheteroalkyl, aryl heteroalkyl, heteroarylheteroalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkoxycycloalkyl, alkoxyheterocycloalkyl, alkoxyaryl, alkoxyheteroaryl, alkoxycarbonyl, alkylaminocarbonyl, alkenyloxy, alkynyloxy, cycloalkyloxy, cycloalkenyloxy, heterocycloalkyloxy, heterocycloalkenyloxy, aryloxy, phenoxy, benzyloxy, heteroaryloxy, arylalkyloxy, arylalkyl, heteroarylalkyl, cycloalkylalkyl, heterocycloalkylalkyl, arylalkyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, alkylsulfinyl, arylsulfinyl, aminosulfinylaminoalkyl, - COOH, -COR^a, -C(O)OR³, CONHR³, NHCOR³, NHCOOR³, NHCONHR³, C(=N0H)R³ -SH, -SR³, -OR³ and acyl.

"Alkyl" as a group or part of a group refers to a straight or branched aliphatic hydrocarbon group, preferably a C₁-C₁₄ alkyl, more preferably C₁-C₁₀ alkyl, most preferably C₁-C₆ unless otherwise noted. Examples of suitable straight and branched C₁-C₆ alkyl substituents include methyl, ethyl, n-propyl, 2-propyl, n-butyl, sec-butyl, t- butyl, hexyl, and the like. The group may be a terminal group or a bridging group.

"Alkylamino" includes both mono-alkylamino and dialkylamino, unless specified. "Mono-alkylamino" means a -NH-Alkyl group, in which alkyl is as defined above. "Dialkylamino" means a -N(alkyl)₂ group, in which each alkyl may be the same or different and are each as defined herein for alkyl. The alkyl group is preferably a C₁- C₆ alkyl group. The group may be a terminal group or a bridging group. "Arylamino" includes both mono-arylamino and di-arylamino unless specified. Mono-arylamino means a group of formula arylNH-, in which aryl is as defined herein, di-arylamino means a group of formula (aryl^N- where each aryl may be the same or different and are each as defined herein for aryl. The group may be a terminal group or a bridging group.

"Acyl" means an alkyl-CO- group in which the alkyl group is as described herein. Examples of acyl include acetyl and benzoyl. The alkyl group is preferably a C₁-C₆ alkyl group. The group may be a terminal group or a bridging group.

"Alkenyl" as a group or part of a group denotes an aliphatic hydrocarbon group containing at least one carbon-carbon double bond and which may be straight or branched preferably having 2-14 carbon atoms, more preferably 2-12 carbon atoms, most preferably 2-6 carbon atoms, in the normal chain. The group may contain a plurality of double bonds in the normal chain and the orientation about each is independently E or Z. Exemplary alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl and nonenyl. The group may be a terminal group or a bridging group.

"Alkoxy" refers to an -O-alkyl group in which alkyl is defined herein. Preferably the alkoxy is a C₁-C₆alkoxy. Examples include, but are not limited to, methoxy and ethoxy. The group may be a terminal group or a bridging group.

"Alkenyloxy" refers to an -O- alkenyl group in which alkenyl is as defined herein. Preferred alkenyloxy groups are C₁-C₆ alkenyloxy groups. The group may be a terminal group or a bridging group.

"Alkynyloxy" refers to an -O-alkynyl group in which alkynyl is as defined herein. Preferred alkynyloxy groups are C₁-C₆ alkynyloxy groups. The group may be a terminal group or a bridging group.

"Alkoxycarbonyl" refers to an -C(O)-O-alkyl group in which alkyl is as defined herein. The alkyl group is preferably a C₁-C₆ alkyl group. Examples include, but not limited to, methoxycarbonyl and ethoxycarbonyl. The group may be a terminal group or a bridging group. "Akylsulfinyl" means a -S(O)-alkyl group in which alkyl is as defined above. The alkyl group is preferably a CrC₆ alkyl group. Exemplary alkylsulfinyl groups include, but not limited to, methylsulfinyl and ethylsulfinyl. The group may be a terminal group or a bridging group.

"Alkylsulfonyl" refers to a -S(O)₂-alkyl group in which alkyl is as defined above. The alkyl group is preferably a C₁-C₆ alkyl group. Examples include, but not limited to methylsulfonyl and ethylsulfonyl. The group may be a terminal group or a bridging group.

"Alkynyl" as a group or part of a group means an aliphatic hydrocarbon group containing a carbon-carbon triple bond and which may be straight or branched preferably having from 2-14 carbon atoms, more preferably 2-12 carbon atoms, more preferably 2-6 carbon atoms in the normal chain. Exemplary structures include, but are not limited to, ethynyl and propynyl. The group may be a terminal group or a bridging group.

"Alkylaminocarbonyl" refers to an alkylamino-carbonyl group in which alkylamino is as defined above. The group may be a terminal group or a bridging group.

"Cycloalkyl" refers to a saturated or partially saturated, monocyclic or fused or spiro polycyclic, carbocycle preferably containing from 3 to 9 carbons per ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like, unless otherwise specified. It includes monocyclic systems such as cyclopropyl and cyclohexyl, bicyclic systems such as decalin, and polycyclic systems such as adamantane. The group may be a terminal group or a bridging group.

"Cycloalkenyl" means a non-aromatic monocyclic or multicyclic ring system containing at least one carbon-carbon double bond and preferably having from 5-10 carbon atoms per ring. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl or cycloheptenyl. The cycloalkenyl group may be substituted by one or more substituent groups. The group may be a terminal group or a bridging group.

The above discussion of alkyl and cycloalkyl substituents also applies to the alkyl portions of other substituents, such as without limitation, alkoxy, alkyl amines, alkyl ketones, arylalkyl, heteroarylalkyl, alkylsulfonyl and alkyl ester substituents and the like.

"Cycloalkylalkyl" means a cycloalkyl-alkyl- group in which the cycloalkyl and alkyl moieties are as previously described. Exemplary monocycloalkylalkyl groups include cyclopropylmethyl, cyclopentylmethyl, cyclohexylmethyl and cycloheptylmethyl. The group may be a terminal group or a bridging group.

"Heterocycloalkyl" refers to a saturated or partially saturated monocyclic, bicyclic, or polycyclic ring containing at least one heteroatom selected from nitrogen, sulfur, oxygen, preferably from 1 to 3 heteroatoms in at least one ring. Each ring is preferably from 3 to 10 membered, more preferably 4 to 7 membered. Examples of suitable heterocycloalkyl substituents include pyrrolidyl, tetrahydrofuryl, tetrahydrothiofuranyl, piperidyl, piperazyl, tetrahydropyranyl, morphilino, 1 ,3- diazapane, 1 ,4-diazapane, 1 ,4-oxazepane, and 1 ,4-oxathiapane. The group may be a terminal group or a bridging group.

"Heterocycloalkenyl" refers to a heterocycloalkyl as described above but containing at least one double bond. The group may be a terminal group or a bridging group.

"Heterocycloalkylalkyl" refers to a heterocycloalkyl-alkyl group in which the heterocycloalkyl and alkyl moieties are as previously described. Exemplary heterocycloalkylalkyl groups include (2-tetrahydrofuryl)methyl,

(2-tetrahydrothiofuranyl) methyl. The group may be a terminal group or a bridging group.

"Heteroalkyl" refers to a straight- or branched-chain alkyl group preferably having from 2 to 14 carbons, more preferably 2 to 10 carbons in the chain, one or more of which has been replaced by a heteroatom selected from S, O, P and N. Exemplary heteroalkyls include alkyl ethers, secondary and tertiary alkyl amines, amides, alkyl sulfides, and the like. The group may be a terminal group or a bridging group. As used herein reference to the normal chain when used in the context of a bridging group refers to the direct chain of atoms linking the two terminal positions of the bridging group. "Aryl" as a group or part of a group denotes (i) an optionally substituted monocyclic, or fused polycyclic, aromatic carbocycle (ring structure having ring atoms that are all carbon) preferably having from 5 to 12 atoms per ring. Examples of aryl groups include phenyl, naphthyl, and the like; (ii) an optionally substituted partially saturated bicyclic aromatic carbocyclic moiety in which a phenyl and a C_5-7 cycloalkyl or C_5-7 cycloalkenyl group are fused together to form a cyclic structure, such as tetrahydronaphthyl, indenyl or indanyl. The group may be a terminal group or a bridging group.

"Arylalkenyl" means an aryl-alkenyl- group in which the aryl and alkenyl are as previously described. Exemplary arylalkenyl groups include phenylallyl. The group may be a terminal group or a bridging group.

"Arylalkyl" means an aryl-alkyl- group in which the aryl and alkyl moieties are as previously described. Preferred arylalkyl groups contain a C_1-5 alkyl moiety. Exemplary arylalkyl groups include benzyl, phenethyl and naphthelenemethyl. The group may be a terminal group or a bridging group.

"Heteroaryl" either alone or part of a group refers to groups containing an aromatic ring (preferably a 5 or 6 membered aromatic ring) having one or more heteroatoms as ring atoms in the aromatic ring with the remainder of the ring atoms being carbon atoms. Suitable heteroatoms include nitrogen, oxygen and sulphur. Examples of heteroaryl include thiophene, benzothiophene, benzofuran, benzimidazole, benzoxazole, benzothiazole, benzisothiazole, naphtho[2,3-b]thiophene, furan, isoindolizine, xantholene, phenoxatine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indole, isoindole, 1 H-indazole, purine, quinoline, isoquinoline, phthalazine, naphthyridine, quinoxaline, cinnoline, carbazole, phenanthridine, acridine, phenazine, thiazole, isothiazole, phenothiazine, oxazole, isooxazole, furazane, phenoxazine, 2-, 3- or 4- pyridyl, 2-, 3-, 4-, 5-, or 8- quinolyl, 1-, 3-, 4-, or 5- isoquinolinyl 1-, 2-, or 3- indolyl, and 2-, or 3-thienyl. The group may be a terminal group or a bridging group.

"Heteroarylalkyl" means a heteroaryl-alkyl group in which the heteroaryl and alkyl moieties are as previously described. Preferred heteroarylalkyl groups contain a lower alkyl moiety. Exemplary heteroarylalkyl groups include pyridylmethyl. The group may be a terminal group or a bridging group. "Lower alkyl" as a group means unless otherwise specified, an aliphatic hydrocarbon group which may be straight or branched having 1 to 6 carbon atoms in the chain, more preferably 1 to 4 carbons such as methyl, ethyl, propyl (n-propyl or isopropyl) or butyl (n-butyl, isobutyl or tertiary-butyl). The group may be a terminal group or a bridging group.

It is understood that included in the family of compounds of Formula (I) are isomeric forms including diastereoisomers, enantiomers, tautomers, and geometrical isomers in "E" or "Z" configurational isomer or a mixture of E and Z isomers. It is also understood that some isomeric forms such as diastereomers, enantiomers, and geometrical isomers can be separated by physical and/or chemical methods and by those skilled in the art.

Some of the compounds of the disclosed embodiments may exist as single stereoisomers, racemates, and/or mixtures of enantiomers and /or diastereomers. All such single stereoisomers, racemates and mixtures thereof, are intended to be within the scope of the subject matter described and claimed.

It is also intended that the present invention extends to, where applicable, solvated as well as unsolvated forms of the compounds. Thus, each formula includes compounds having the indicated structure, including the hydrated as well as the non-hydrated forms.

The compounds of the various embodiments include pharmaceutically acceptable salts, prodrugs, N-oxides and active metabolites of such compounds, and pharmaceutically acceptable salts of such metabolites.

The term "pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the above-identified compounds, and include pharmaceutically acceptable acid addition salts and base addition salts. Suitable pharmaceutically acceptable acid addition salts of compounds of Formula (I) may be prepared from an inorganic acid or from an organic acid. Examples of such inorganic acids are hydrochloric, sulfuric, and phosphoric acid. Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, heterocyclic carboxylic and sulfonic classes of organic acids, examples of which are formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, fumaric, maleic, alkyl sulfonic, arylsulfonic. Suitable pharmaceutically acceptable base addition salts of compounds of Formula (I) include metallic salts made from lithium, sodium, potassium, magnesium, calcium, aluminium, and zinc, and organic salts made from organic bases such as choline, diethanolamine, morpholine. Other examples of organic salts are: ammonium salts, quaternary salts such as tetramethylammonium salt; amino acid addition salts such as salts with glycine and arginine. Additional information on pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Co., Easton, PA 1995. In the case of agents that are solids, it is understood by those skilled in the art that the inventive compounds, agents and salts may exist in different crystalline or polymorphic forms, all of which are intended to be within the scope of the present invention and specified formulae.

"Prodrug" means a compound which is convertible in vivo by metabolic means (e.g. by hydrolysis, reduction or oxidation) to a compound of formula (I). For example an ester prodrug of a compound of formula (I) containing a hydroxyl group may be convertible by hydrolysis in vivo to the parent molecule. Suitable esters of compounds of formula (I) containing a hydroxyl group, are for example acetates, citrates, lactates, tartrates, malonates, oxalates, salicylates, propionates, succinates, fumarates, maleates, methylene-bis-β-hydroxynaphthoates, gestisates, isethionates, di-p-toluoyltartrates, methanesulphonates, ethanesulphonates, benzenesulphonates, p-toluenesulphonates, cyclohexylsulphamates and quinates. As another example an ester prodrug of a compound of formula (I) containing a carboxy group may be convertible by hydrolysis in vivo to the parent molecule. (Examples of ester prodrugs are those described by FJ. Leinweber, Drug Metab. Res., 18:379, 1987).

Compounds as defined supra are for modulating (and particularly upregulating) protein serine/threonine phosphatase activity. In the context of the present invention the term "protein serine/threonine phosphatase activity" and the like, as used herein includes any function(s) exhibited or performed by the protein serine/threonine phosphatase that is ascribed to the naturally occurring form of the enzyme as measured or observed in vivo {i.e., in the natural physiological environment of the enzyme) or in vitro (i.e., under laboratory conditions). Studies have shown that the catalytic domains of these phosphatases have a high degree of identity. These phosphatases include two families namely: phospho protein phosphatase (PPP) and protein phosphatase magnesium dependent (PPM). There are several isoforms of protein serine/threonine phosphatases, comprising PP1 , PP2A, PP2B, PP2C, PP4, PP5, PP6 and PP7, as well as their various isoforms. Clinically relevant phosphatases include PP1 α, PP1 β, PP1γ1 , PP1γ2, PP2A, PP2B (calcineurin), PP2C, PP4, PP5 and PP6. PP2A and PP5 are particularly important members, being associated with a number of pathology in humans.

In identifying the compounds of the present invention, the applicant has defined binding motifs on the surface of phosphatases. By way of overview, rationalized candidate compound optimization studies have been performed using computer- based modelling of sulphate and selenate in complex with the protein serine/threonine phosphatase PP2A and in complex with the protein serine/threonine phosphatase PP5. Molecular interactions were analysed and a number of binding motifs implicated in the modulation of protein serine/threonine phosphatase activity identified. Examples of so identified motifs follow:

(a) D57, H59, D85, R89, N1 17, H1 18, H167, R214, H241 ;

(b) D57, H59, D85, N117, H167, H241 ; (C) 162,163,235,236,250,254,278,282,284;

(d) N1 17,H1 18,S120,R121 ,Q122,l 123,T124,E188,V189,P190,H191 ,G192,C196, W200,A216,G217;

(e) I 14,N18,M66,F69,R7O,G73,K74,S75,Y8O,L99,L1 O3;

(f) F164,L166,G169,L17O,S171 ,I 174,I 18O,L198,W2O9,F228; (g) L39,Y86,V97,V101 ,l1 13,E1 19,F146,L149,F150,L153;

(h) Y86,T96,V97,L100,V101 ,l 1 13,F150,L153;

(i) Q46,E47,V48,R49,P51 ,V52,N79,Y80,L81 ,K104,R108,E109,R1 10,11 1 1.T1 12;

C) L31 ,A35,L39,L100,V101 ,K104,V105,l1 13,L149,L153;

(k) D57,V58,Q61 ,D64,L65,L68,S261 ,A262,P263,F289; (I) C55,G56,D57,V58,H59,L81 ,F82,M83,G84,D85,Y86,L100,L1 14,S261 ;

(m) D57,H59,D85,R89,N1 17,H1 18,l 123,Y127,W200,R214,H241 ,F260,Y265;

(n) V28,K29,C32,E33,K36,K144,Y145,D148,L149;

(o) I 14,S75,P76,L1 O3,R1 O6,Y1 O7,I1 1 1 ;

(P) L39,T40,D151 ,Y152,L153,R185,L186,Q187; (q) H191 ,E192,G193,C196,D197,A216,G217,Y218; (r) Y107,R108,E109,R110;

(s) P51 ,V52,T53,D77,T78,N79,Y80,M276,E277,L278,D279 ;

(t) D131 ,L134,R135,Y307,F308;

(u) V244,M245,E246,G247,Y248,N249,W250; (v) W200,S201 ,D202,S212,P213,R214,A216,G217,Y218,T219,F220;

(w) R89,R214,H241 ,Q242,L243,F260,Y265;

(X) F6,W13,S30,L31 ,E33,K34,E37;

(y) L10,D1 1 ,l14,E15,P76,R106,Y107;

(Z) G90,Y91 ,Y92,S93,G128,F129,D131 ,E132,R135,K136; (aa) L17O,S171 ,I174,D175,W2O9,I224,T227,F228;

(bb) H59,D85,D88,R89,H1 18,Y127,Y265;

(CC) Y92, Y267, K294, R295, G296, E297, P298,V308;

(dd) P203,D204,D205,R206,G207,G221 ,Q222,D223,Q242,N249,C251 ,H252,D253;

(ee) L183,D184,R185,Q187,P190,P194,H195; (ff) Y267,P291 ,A292,R294;

(gg) P203, R239,Q242, L243,V244, N249.T258;

(hh) E67.I71 ,F289,D290,P291 ,A292,P293;

(ii) E33,K36,E37;

Cj) V5,F6,T7,K8,E9; (kk) I21 1 ,S212,G215,A216,G217,Y218;

(II) E226,N229,H230,L234,N255;

(mm) T176,L177,D178,R181 ,N232;

(nn) H63,H66,E67,R70,A292,P293,R294;

(oo) H63,Y92,Y267,R294,R295; (PP) D202,P203,D204,P213,T219,F220,Q242;

(qq) V300,R303,T304,P305;

(rr) H63,P293,R294,R295;

(SS) T40,N44,L183,D184,R185,L186;

(tt) E42,Q46,V48,K104,R108,E109,l1 11 ,T112; (UU) D204,R206,G210,l21 1 ,S212,P213,T219;

(W) D204,R206,l211 ,P213,T219; and

(WW) N18,E19,C20,F62,H62,M63.

(XX) R89, N117, H1 18, 1123, Y127, W200, P213, R214, H241 , Q242, L243, F260,

Y265; (yy) N1 17, S120, R121 , Q122, 1123, Y127, E188, V189, P190, H191 , W200;

(ZZ) D204, D205, C221 , Q222, G251 , H252, D253; (aaa) R89, R214;

(bbb) H191.Q122;

(CCC) E188, R121;

(ddd) H252, D253; and (eee) D290.

Given the identification of the binding motifs, X-ray structure coordinates were generated to identify the relative positions of the atoms in the binding motifs. Such relative data obtained does not, however, define an absolute set of points in space. As would be understood by a skilled addressee a set of structure coordinates for any crystal structure defines a relative set of points that, in turn, define a configuration of atoms in three dimensions. In essence the importance of crystal structure data is that it provides information as to the spatial relationship of the atoms in the crystal with respect to each other. An exemplary crystal structure and co-ordinates are disclosed in Cho and Xu, "Crystal structure of a protein phosphatase 2A heterotrimeric holoenzyme" Nature 445:53-57, 2007 and as publicly disclosed in the RCSB Protein Databank under accession 2iae (http://www.rcsb.org/pdb/files/2iae.pdb); as accessed

7 December 2007; the contents of both being herein incorporated by reference.

The Applicant has shown that selenate can bind to the catalytic subunit of a protein serine/threonine phosphatase and has been shown to modulate (i.e., enhance) the activity of a protein serine/threonine phosphatases such as PP2A. Therefore, identification and/or design of compounds that mimic, enhance, disrupt or compete with the interactions of selenate with the catalytic subunit of a protein serine/threonine phosphatase are also desirable and thus form another aspect of the present invention.

Traditionally drug development and design has been performed by way of large scale screening programs, by the use of structure/activity studies, or by serendipity. Whilst these techniques are successful to a point they are labor intensive and rely on luck and the breadth of the study rather than rigorous principles of the desired interaction at a molecular level. More recently drug design has been carried out by using computer assisted models which can model the desired interaction hopefully leading to improved therapeutic compounds. Computers therefore provide the drug developer with the ability to screen, identify, select and/or design chemical entities capable of associating the particular receptor of interest. As would be clear, however, before this can be carried out, detailed knowledge of the structure of the receptor must be known so that this data can be input into the computer software that carries out the modelling. Detailed knowledge of the exact structural coordinates of a receptor permits the design and/or identification of synthetic compounds and/or other molecules which are spatially adapted for optimal interaction with the binding site of the receptor. Applicant has identified the relevant coordinates allowing for computer aided drug design can be used to identify or design candidate compounds, such as inhibitors, agonists and antagonists that bind to and/or modulate the activity of a protein serine/threonine phosphatase. Once identified and screened for binding capability and/or biological activity, these inhibitors/agonists/antagonists may be used therapeutically or prophylactically to block the receptor and therefore should be useful in treating a condition associated with the activity of a protein/serine phosphatase.

Candidate compounds that are identified or designed by computer methods to interact with one or more of the binding motifs identified on the catalytic subunit of a protein serine/threonine phosphates are potential modulators of the enzyme's activity and are therefore potential drug candidates. It has therefore been possible to identify potential candidate compounds by consideration of the X-Ray crystallography data. Such data can be used in computational methods well known in the art to determine which residues are on the surface of the catalytic subunit representing the one or more binding motifs identified by the inventors and therefore potentially able to interact with other molecules in solution. In general, these techniques rely on graphic representations of the receptor and computer assisted manipulation of the graphic representation. The structural coordinate data stored in a machine-readable storage medium that is capable of displaying a graphical three-dimensional representation of the structure of the binding motif(s) or portion(s) thereof in a liganded and unliganded state can therefore be used in drug discovery. The structure coordinates of the candidate compound are used to generate a three-dimensional image that can be computationally fitted to the three-dimensional image of the enzyme, or a fragment thereof.

Once a suitable candidate compound or fragments thereof have been selected, they can be assembled into a single compound or complex. Assembly may be preceded by visual inspection of the relationship of the fragments to each other on the three- dimensional image displayed on a computer screen in relation to the structure coordinates of the target compound or site. This would be followed by manual model building using software such as Quanta or Sybyl. Once a compound has been designed or selected by such methods, the efficiency with which that compound can bind to a target site may be tested and optimized by computational evaluation. For example, an effective ligand will preferably demonstrate a relatively small difference in energy between its bound and free states; i.e. a small deformation energy of binding. Thus the most efficient ligand should preferably be designed with a deformation energy of binding of not greater than about 10kcal/mole, preferably, not greater than 7 kcal/mole. Ligands may interact with the target in more than one conformation that is similar in overall binding energy. In those cases, the deformation energy of binding is taken to be the difference between the energy of the free entity and the average energy of the conformations observed when the inhibitor binds to the protein.

A candidate compound designed or selected as binding to a target may be further computationally optimized so that in its bound states it would preferably lack repulsive electrostatic interaction with the target enzyme. Such non-complementary (e.g., electrostatic) interactions include repulsive charge-charge, dipole-dipole and charge- dipole interactions. Specifically, the sum of all electrostatic interactions between the ligand and the target, when the ligand is bound to the target, preferably makes a neutral or favourable contribution to the enthalpy of binding.

Specific computer software is available in the art to evaluate compound deformation energy and electrostatic interaction. Examples of programs designed for such uses include: Gaussian 92, revision C [MJ. Frisch, Gaussian, Inc., Pittsburgh, Pa. COPYRGT. 1992]; AMBER, version 4.0 [P.A. Kollman, University of California at San Francisco, COPYRGT.1994]; QUANTA/CHARMM [Molecular Simulations, Inc., Burlington, Mass. COPYRGT.1994]; and Insight Il/Discover (Biosysm Technologies Inc., San Diego, Calif. COPYRGT.1994). These programs may be implemented, for instance, using a Silicon Graphics workstation, IRIS 4D/35 or IBM RISC/6000 workstation model 550. Other hardware systems and software packages will be known to those skilled in the art.

Once the candidate compound has been optimally selected or designed, as described above, substitutions may then be made in some of its atoms or side groups in order to improve or modify its binding properties. Generally initial substitutions are conservative, i.e., the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. It should, of course, be understood that components known in the art to alter conformation should be avoided. Such substituted chemical compounds may then be analyzed for efficiency of fit to the desired target site by the same computer methods described in detail, above. Again, all these facts are familiar to the skilled person.

Another approach is the computational screening of small molecule data bases for chemical entities or compounds which can bind in whole, or in part, to a desired target. In this screening, the quality of fit of such entities to the binding site may be judged either by shape complementarity or by estimated interaction energy.

The computational analysis and design of molecules, as well as software and computer systems therefore, are described in U. S Patent No. 5,978,740 which is included herein by reference, comprising specifically, but not by way of limitation, the computer system diagram described with reference to and illustrated in Figure 3 thereof, as well as the data storage media diagram described with reference to and illustrated in Figure 4s and 5 thereof.

Pursuant to the present invention, such stereochemical complementarity is characteristic of a molecule which matches intra-site surface residues lining the binding regions identified herein. By "match" it is meant that the identified portions interact with the surface residues, for example, via hydrogen bonding or by enthalpy/entropy-reducing Van der Waals interactions which promote desolvation of the biologically active compound within the site, in such a way that retention of the biologically active compound within the groove is energetically favoured.

In general, the design of a molecule possessing stereochemical complementarity can be accomplished by means of techniques which optimize, either chemically or geometrically, the "fit between a molecule and a target receptor. Suitable such techniques are known in the art. (See Sheridan and Venkataraghavan, 1987; Goodford 1984; Beddell 1985; HoI, 1986; and Verlinde 1994, the respective contents of which are hereby incorporated by reference. See also Blundell 1987).

Thus, there are at least two approaches in determining whether a compound complements the shape of a binding site on the protein phosphatase, and can therefore upregulate the activity of the protein phosphatase. In the first of these, the geometric approach, the number of internal degrees of freedom, and the corresponding local minima in the molecular conformation space, is reduced by considering only the geometric (hard-sphere) interactions of two rigid bodies, where one body (the active site) contains "pockets" or "grooves" which form binding sites for the second body (the complementing molecule, as ligand). The second approach entails an assessment of the interaction of different chemical groups ("probes") with the binding motif(s) of the present invention at sample positions within and around the site, resulting in an array of energy values from which three-dimensional contour surfaces at selected energy levels can be generated.

The geometric approach is illustrated by Kuntz et al. (Kuntz, I. D. et al. J. MoI. Biol. 161 :269-288, 1982; the contents of which are hereby incorporated by reference), whose algorithm for the design of candidate compounds {e.g., ligands) is implemented in a commercial software package distributed by the Regents of the University of California and further described in a document, provided by the distributor, entitled "Overview of the DOCK Package, Version 1.0,", the contents of which are hereby incorporated by reference. Pursuant to the Kuntz algorithm, the shape of the cavity represented by the substrate/ligand binding site is defined as a series of overlapping spheres of different radii. One or more extant databases of crystallographic data, such as the Cambridge Structural Database System maintained by Cambridge University (University Chemical Laboratory, Lensfield Road, Cambridge CB2 1 EW, U.K.) and the Protein Data Bank maintained by the Research Collaboratory for Structural Bioinformatics (RCSB; http//www.rcsb.org/index.html) is then searched for molecules which approximate the shape thus defined.

Candidate compounds identified in this way, on the basis of geometric parameters, can then be modified to satisfy criteria associated with chemical complementarity, such as hydrogen bonding, ionic interactions and van der Waals interactions.

The chemical-probe approach to ligand design is described, for example, by Goodford {J. Med. Chem., 28:849-857, 1985; the contents of which are hereby incorporated by reference), and is implemented in several commercial software packages, such as GRID (product of Molecular Discovery Ltd., West Way House, Elms Parade, Oxford OX2 9LL, U.K.). Pursuant to this approach, the chemical prerequisites for a site- complementing molecule are identified at the outset, by probing the substrate/ligand binding site with different chemical probes, e.g., water, a methyl group, an amine nitrogen, a carboxyl oxygen, and a hydroxyl. Favoured sites for interaction between the active site and each probe are thus determined, and from the resulting three- dimensional pattern of such sites a putative complementary molecule can be generated.

Programs suitable for searching three-dimensional databases to identify molecules bearing a desired pharmacophore include: MACCS-3D and ISIS/3D (Molecular Design Ltd., San Leandro, CA), ChemDBS-3D (Chemical Design Ltd., Oxford, U.K.), and Sybyl/3DB Unity (Tripos Associates, St. Louis, MO).

Programs suitable for pharmacophore selection and design include: DISCO (Abbott Laboratories, Abbott Park, IL), Catalyst (Bio-CAD Corp., Mountain View, CA), and ChemDBS-3D (Chemical Design Ltd., Oxford, U.K.). Databases of chemical structures are available from a number of sources including Cambridge Crystallographic Data Centre (Cambridge, U.K.) and Chemical Abstracts Service (Columbus, OH).

De novo design programs include Ludi (Biosym Technologies Inc., San Diego, CA), Sybyl (Tripos Associates) and Aladdin (Daylight Chemical Information Systems, Irvine, CA).

In one embodiment of the compound, the compound has a high affinity for the selected target site (i.e., binding motif). The affinity constant is preferably <1 μM, more preferably < 1 nM.

In vivo or in vitro methods may be used to confirm the results of in silico screening. In one embodiment, a cell-based assay is conducted under conditions which are effective to screen for candidate compounds useful in the method of the present invention. Effective conditions include, but are not limited to, appropriate media, temperature, pH and oxygen conditions that permit the growth of the cell that expresses the receptor. An appropriate, or effective, medium refers to any medium in which a cell that naturally or recombinantly expresses a catalytic subunit of a protein serine/threonine phosphatase, when cultured, is capable of cell growth and expression of the catalytic subunit. Such a medium is typically a solid or liquid medium comprising growth factors and assimilable carbon, nitrogen and phosphate sources, as well as appropriate salts, minerals, metals and other nutrients, such as vitamins. Culturing is carried out at a temperature, pH and oxygen content appropriate for the cell. Such culturing conditions are within the expertise of one of ordinary skill in the art.

Cells that are useful in the cell-based assays of the present invention include any cell that expresses a catalytic subunit of a protein serine/threonine phosphatase and particularly, other proteins that provide signal transduction cascades associated with protein serine/threonine phosphatases. Such cells include, but are not limited to, red blood cells, neurons, endothelial cells, epithelial cells, lymphocytes and fibroblasts. Additionally, certain cells may be induced to express a catalytic subunit of a protein serine/threonine phosphatase, for example, some tumour cells. Therefore, cells that express a catalytic subunit of a protein serine/threonine phosphatase can include cells that naturally express the catalytic subunit, recombinantly express the catalytic subunit, or which can be induced to express catalytic subunit.

The assay of the present invention can also be a non-cell based assay. In this embodiment, the candidate compound can be directly contacted with an isolated catalytic subunit of a protein serine/threonine phosphatase, or a fragment thereof, and the ability of the candidate compound to bind to the enzyme subunit can be evaluated, such as by an immunoassay or other binding assay. The assay can, if desired, additionally include the step of further analyzing whether candidate compounds which bind to a portion of the receptor are capable of increasing or decreasing the activity of the protein serine/threonine phosphatase. Such further steps can be performed by cell-based assay, as described above, or by non-cell-based assay.

Alternatively, a soluble catalytic subunit of a protein serine/threonine phosphatase may be recombinantly expressed and utilized in non-cell based assays to identify compounds that bind to it. Recombinantly expressed catalytic subunits or fusion proteins containing one or more extracellular domains of the catalytic subunit can be used in the non-cell based screening assays. In non-cell based assays, the recombinantly expressed catalytic subunit can be attached to a solid substrate by means well known to those in the art. For example, the catalytic subunit and/or cell lysates containing such subunits can be immobilized on a substrate such as artificial membranes, organic supports, biopolymer supports and inorganic supports. The protein can be immobilized on the solid support by a variety of methods including adsorption, cross-linking (including covalent bonding), and entrapment. Adsorption can be through Van del Waal's forces, hydrogen bonding, ionic bonding, or hydrophobic binding. Exemplary solid supports for adsorption immobilization include polymeric adsorbents and ion-exchange resins. Solid supports can be in any suitable form, including in a bead form, plate form, or well form. The test compounds are then assayed for their ability to bind to the catalytic subunit.

In one embodiment, a BIAcore machine can be used to determine the binding constant of a complex between the catalytic subunit and a ligand {e.g., sodium selenate) in the presence and absence of the candidate compound. The dissociation constant for the complex can be determined by monitoring changes in the refractive index with respect to time as buffer is passed over the chip (see O'Shannessy et al. Anal. Biochem. 212:457-468, 1993; and Schuster et al., Nature 365:343-347, 1993).

Other suitable assays for measuring the binding of a candidate compound to a catalytic subunit of a protein serine/threonine phosphatase, and or for measuring the ability of a candidate compound to affect the binding of a catalytic subunit of a protein serine/threonine phosphatase to a ligand such as sodium selenate include, for example, immunoassays such as enzyme linked immunoabsorbent assays (ELISA) and radioimmunoassays (RIA), as well as cell-based assays comprising, cytokine secretion assays, or intracellular signal transduction assays that determine, for example, protein or lipid phosphorylation.

In another aspect the present invention provides a method for upregulating the activity of a serine/threonine protein phosphatase, the method comprising the step of exposing the cell to an effective amount of a compound or composition as described herein. In one embodiment of the method the protein serine/threonine phosphatase is selected from the group consisting of PP1 α, PP1 β, PP1γ1 , PP1γ2, PP2A, PP2B (calcineurin), PP2C, PP4, PP5 and PP6. In one form, the protein serine/threonine phosphatase is PP2A. These methods may be performed in vivo or in vitro, however preferably they are performed in vitro. For example, the compounds may be utilised to in a research context to ascertain further effects of protein phosphatase upregulation.

The skilled person will be able to decide whether any compound described herein has the ability to upregulate the activity of a protein phosphatase. An exemplary method may include contacting the compound with a protein serine/threonine phosphatase, or with an analogue, a homolog or a fragment thereof, and determining the ability of the compound to modulate protein serine/threonine phosphatase activity.

Alternatively, protein serine/threonine phosphatase activity is determined by measuring the level of enzyme present in cells by Western blotting. For example, protein levels of the enzyme can be measured in fibroblasts using an antibody {e.g., an anti-PP2A antibody; Biosource). Levels of a different protein can also be measured in the same sample as a reference protein for normalization. Examples of possible reference proteins include, but are not limited to, annexin-ll or actin. In another embodiment, the level of protein serine/threonine phosphatase activity in AD and AC cells is assayed according to a procedure (Pierce Biotechnology) using p-nitrophenyl phosphate (PNPP) as the substrate. For instance, the enzyme activity assays are carried out in a 96-well microplate. The reaction is initiated by adding about 10 μl of each AC or AD cell lysate into about 90 μl of reaction mixture, incubated at about 30⁰C for about 15 minutes, and measured in a BioRad microplate reader at a wavelength of 420 nM. After subtraction of values from reactions in which about 10 nM of an enzyme inhibitor such as okadiac acid is present, the activity of the enzyme is calculated according to a standard curve produced by a series of known concentrations of purified enzyme.

Compounds capable of upregulating (i.e. agonizing) protein phosphatase activity may exhibit improved binding to a catalytic subunit of a protein serine/threonine phosphatase when compared with the ability of a natural ligand of the enzyme, and also include compounds that enhance the binding of a natural ligand to the catalytic subunit of the enzyme. Agonists may also be identified by their ability to: (1 ) bind to, or otherwise interact with, a catalytic subunit of a protein serine/threonine phosphatase at a higher level than, for example, a natural ligand of the enzyme and/or; (2) enhance binding of the catalytic subunit of the enzyme to its natural ligand. Another suitable agonist compound of the present invention can include a compound that binds to a catalytic subunit of a protein serine/threonine phosphatase in the absence of a natural ligand of the enzyme in such a manner that enzyme-mediated cellular signal transduction is stimulated.

Once a compound has been identified as capable of modulating the activity of protein serine/threonine phosphatase, as herein described, the compound can administered to a subject to treat a condition associated with the activity of a protein serine/threonine phosphatase. Accordingly, a further aspect of the invention provides a method for treating a condition associated with the activity of a protein serine/threonine phosphatase in a subject, the method comprising the administration to a subject in need thereof an effective amount of a compound described herein. It will be understood that the compounds described by the present invention need not necessarily be administered for the purpose of restoring the activity of a phosphatase having a lower than normal level of phosphatase activity up to a level that is normal, or closer to normal, or higher than normal. The compounds may be useful in conditions whereby the activity of the phosphatase is normal, but enhancement of activity to a level higher than normal has a beneficial effect. As the skilled artisan understands (and as discussed in the Background section herein), the level of phosphorylation of a given protein may be dependent on the balance between kinase activity and phosphatase activity in that cell. Thus, for example, where a condition is the result of excessive kinase activity (leading the a higher than desired level of phosphorylation of a given protein), it may be desirable to upregulate the activity of a phophatase to a level greater than normal to return the level of phosphorylation to a level closer to normal.

In certain embodiments of the method, the condition is associated with low phosphatase activity. Accordingly, in another aspect the present invention provides a method for treating or preventing a condition associated with the activity of a serine/threonine protein phosphatase, the method comprising administering to a subject in need thereof a therapeutically effective amount of a composition as described herein.

In one embodiment of the method, the protein serine/threonine phosphatase is selected from the group consisting of PP1 α, PP1 β, PP1γ1 , PP1γ2, PP2A, PP2B (calcineurin), PP2C, PP4, PP5 and PP6. It will be understood that the subject may be any mammal, including any non-human mammal. However, in a preferred form of the method the subject is a human.

Generally speaking, the effective amount will vary depending on the health and physical condition of the individual to be treated, the taxonomic group of the individual to be treated, and the formulation of the composition, the assessment of the medical situations and other relevant factors. It is expected that the amount will fall within a relatively broad range that can be determined through routine trials. In one form of the method, the method, the condition associated with the activity of a serine/threonine protein phosphatase is a neurological disease, or more particularly a neurodegenerative disease. As used herein, the term "neurological disorder" means a disorders that affects the central nervous system, the peripheral nervous system or the autonomic nervous system. The term "neurodegenerative disease" includes a neurological disease characterised by loss or degeneration of neurons. Neurodegenerative diseases include neurodegenerative movement disorders and neurodegenerative conditions relating to memory loss and/or dementia. In one form of the method the neurodegenerative diseases is a tauopathy. In general tauopathies are considered to be a group of dementias and movement disorders which have as a common pathological feature, the presence of intracellular aggregations of abnormal filaments of tau protein. The tau protein in the aggregations may be hyperphosphorylated tau. These aggregations of tau protein filaments in tauopathies can be identified by standard diagnostic techniques such as staining and light microscopy. In contrast, non-tauopathy neurological disorders, some of which while associated with aberrant tau protein, such as hyperphosphorylated tau protein, or to an abnormal amount of tau protein, do not display intracellular aggregations of abnormal tau. It is to be understood, however, that the present methods extend to tauopathies and non-tauopathies.

In another form of the method, the neurodegenerative disease is a α-synucleopathy. The term "α-synucleopathy" as used herein, unless otherwise stated, refers to a disease characterised by the presence of pathological deposition of insoluble α- synuclein polymers or aggregates intracellular and/or extracellularly.

Examples of neurodegenerative diseases include, but are not limited to, presenile dementia, senile dementia, Alzheimer's disease, Parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy (PSP), Pick's disease, primary progressive aphasia, frontotemporal dementia, corticobasal dementia, Parkinson's disease, dementia with Lewy bodies, Down's syndrome, multiple system atrophy, amyotrophic lateral sclerosis (ALS) and Hallervorden-Spatz syndrome.

In some embodiments, the neurological disease is selected from the group consisting of Creutzfeldt-Jakob disease, Huntington's disease, stroke, cerebral ischaemia, dementia associated with stroke or cerebral ischaemia, dementia associated with HIV, disorders associated with excitotoxicity, epilepsy, seizures, schizophrenia, bipolar disorder, depression, mood disorder, multiple sclerosis, acute brain trauma (severe traumatic brain injury) and motor neurone disease.

In one embodiment of the method the neurological disease is a disorder associated with excitotoxicity. As used herein, the term "disorders associated with excitotoxicity" are disorders that involve excessive activation of glutamate receptors in the brain, Disorders associated with excitotoxicity include, ischaemia during stroke, trauma, hypoxia, hypoglycaemia and hepatic encephalopathy; disorders related to long term plastic changes in the central nervous system such as chronic pain, drug tolerance, drug dependence, drug addiction and tardive dyskinesia, epilepsy, schizophrenia anxiety, depression, acute pain and tinnitis,

In another form of the method, the condition associated with the activity of a serine/threonine protein phosphatase is a hyperproliferative disorder. The term

"hyperproliferative disorder" includes conditions involving excess cell proliferation, relative to that occurring with the same type of cell in the general population and/or the same type of cell obtained from a patient at an earlier time. The term denotes malignant as well as non-malignant cell populations. Such disorders have an excess cell proliferation of one or more subsets of cells, which often appear to differ from the surrounding tissue both morphologically and genotypically. The excess cell proliferation can be determined by reference to the general population and/or by reference to a particular patient, e.g. at an earlier point in the patient's life.

Hyperproliferative cell disorders can occur in different types of animals and in humans, and produce different physical manifestations depending upon the affected cells.

Hyperproliferative cell disorders include cancers; blood vessel proliferative disorders such as restenosis, atherosclerosis, in-stent stenosis, vascular graft restenosis, etc.; fibrotic disorders; inflammatory disorders, e.g. arthritis, etc.; endometriosis; benign growth disorders such as prostate enlargement and lipomas; and autoimmune disorders. Cancers of particular interest include carcinomas, e.g. colon, prostate, breast, melanoma, ductal, endometrial, stomach, dysplastic oral mucosa, invasive oral cancer, non-small cell lung carcinoma, transitional and squamous cell urinary carcinoma, etc.; neurological malignancies, e.g. neuroblastoma, gliomas, etc.; hematological malignancies, e.g. childhood acute leukaemia, non-Hodgkin's lymphomas, chronic lymphocytic leukaemia, malignant cutaneous T-cells, mycosis fungoides, non-MF cutaneous T-cell lymphoma, lymphomatoid papulosis, T-cell rich cutaneous lymphoid hyperplasia, bullous pemphigoid, discoid lupus erythematosus, lichen planus, etc.; sarcomas, melanomas, adenomas; benign lesions such as papillomas, and the like, uterine, testicular and ovarian carcinomas, endometriosis, squamous and glandular epithelial carcinomas of the cervix.

Other hyperproliferative disorders that may be associated with altered activity of phosphorylation modifying enzyme(s) include a variety of conditions where there is proliferation and/or migration of smooth muscle cells, and/or inflammatory cells into the intimal layer of a vessel, resulting in restricted blood flow through that vessel, i.e. neointimal occlusive lesions. Occlusive vascular conditions of interest include atherosclerosis, graft coronary vascular disease after transplantation, vein graft stenosis, peri-anastomatic prosthetic graft stenosis, restenosis after angioplasty or stent placement, and the like.

Other disorders and conditions of interest relate to epidermal hyperproliferation, tissue remodelling and repair. For example, the chronic skin inflammation of psoriasis is associated with hyperplastic epidermal keratin ocytes.

Other disorders of interest include inflammatory disorders and autoimmune conditions comprising psoriasis, rheumatoid arthritis, multiple sclerosis, scleroderma, systemic lupus erythematosus, Sjogren's syndrome, atopic dermatitis, asthma, and allergy. Target cells susceptible to the treatment include cells involved in instigating autoimmune reactions as well as those suffering or responding from the effects of autoimmune attack or inflammatory events, and include lymphocytes and fibroblasts.

One particular hyperproliferative disease to which the compounds of the present invention are applicable is cancer. The cancer may be selected from the group consisting of brain, lung, liver, spleen, kidney, lymph node, small intestine, pancreas, blood cells, colon, stomach, breast, endometrium, prostate, testicle, ovary, skin, head and neck, esophagus, bone marrow and blood cancer. In more specific embodiments, the cancer is a lung cancer, colon cancer, breast cancer or cervical cancer.

Other hyperproliferative disorders are selected from benign adenoma tumor cells, angiofibroma tumor cells, hemangioma tumor cells, leiomyoma tumor cells (fibroid), benign chorangioma tumor cells, cystadenoma tumor cells, dermoid tumor cells, desmoid tumor cells, fibroadenoma tumor cells, fibroma tumor cells, benign ganglioneuroma tumor cells, lipoma tumor cells, meningioma tumor cells, myxoma tumor cells, neurofibroma tumor cells, nevus tumor cells, osteochondroma tumor cells, pheochromocytoma tumor cells, polyposis tumor cells, schwannoma tumor cells, benign teratoma tumor cells, benign thymoma tumor cells and Brenner tumor cells. In specific embodiments, the tumor cells are prostatic intraepithelial neoplasia (PIN) tumor cells, benign prostatic hyperplastic (hypertrophic) tumor cells, ductal carcinoma in situ (DCIS) tumor cells, meningioma tumor cells or benign colonic tumor cells (colon polyps).

In another embodiment of the method, the condition associated with the activity of a serine/threonine protein phosphatase is a non-tumor disease or disorder associated with aberrant angiogenesis. As used herein, the term "non-tumor disease or disorder associated with aberrant angiogenesis" refers to diseases or disorders that are associated with abnormal or excessive angiogenesis that do not involve benign or malignant tumors. For the avoidance of doubt, the term "angiogenesis" as used herein refers to the physiological process of growing new blood vessels from preexisting blood vessels. Angiogenesis is required in normal growth processes and to assist healing of wounds. The term "aberrant angiogenesis" refers to abnormal angiogenesis where new blood vessels are produced, or over-produced in inappropriate situations. For example, in exudative age-related macular degeneration, vision loss is at least in part due to excessive blood vessel growth in the choriocapillaris resulting in blood and protein leakage below the retina.

For example, non-tumor diseases or disorders associated with aberrant angiogenesis include conditions improved by removal, inhibition or reduction of angiogenic growth factors. As used herein, the term "angiogenic growth factor" refers to a factor that boosts or stimulates angiogenesis. Many angiogenic factors are peptides or proteins that bind to receptors as part of a signalling pathway. Angiogenenic fagtors include, but are not limited to, vascular endothelial growth factors (VEGF), angiogenin, angiopoietin-1 , Del-1 and fibroblast growth faotors (FGF) such as acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF). Such disease and disorders include clinical conditions characterised by excessive vascular endothelial cell proliferation, vascular permeability, edema or inflammation comprising macular degeneration, especially age-related macular degeneration comprising wet macular degeneration and dry macular degeneration; retinopathies such as diabetic retinopathy, ischaemic retinal vein occlusion and retinopathy of prematurity; endometriosis, restenosis, psoriasis and rheumatoid arthritis, brain edema associated with injury, stroke or tumor, edema associated with inflammatory conditions such as psoriasis, and arthritis comprising rheumatoid arthritis, asthma, generalised edema associated with burns, ascites and pleural effusion, especially macular degeneration, diabetic retinopathy, endometriosis and restenosis.

The method of treating tumor diseases or disorders associated with aberrant angiogenesis includes the administration of a therapeutically effective amount of a composition as described herein. As used herein, the term "therapeutically effective amount" in the context of treating or preventing a non-tumor disease or disorder associated with aberrant angiogenesis is meant the administration of an amount of composition, either in a single dose or as part of a series of doses, that is effective for inhibiting the activity of one or more angiogenic factors and/or inhibiting neovascularisation.

In one form of the method, the therapeutically effective amount is an angiogenic factor-inhibiting amount". The term "angiogenic factor-inhibiting amount" is an amount of composition that blocks or reduces the activity of an angiogenic factor. For example, the angiogenic factor-inhibiting amount may be an amount effective at inhibiting or reducing the effects of VEGF. The activity of VEGF may be blocked or reduced by compounds or agents that prevent VEGF binding to its receptor by binding to VEGF or by competitively binding to VEGFR, inhibition of receptor tyrosine kinases preventing signal transduction in angiogenesis. Similarly the angiogenic factor- inhibiting amount may block or reduce the activity of other angiogenic factors such as angiogenin, angiopoietin-1 , DeI-I and FGF.

A composition to be administered to a patient, such as in this embodiment, generally includes the compound identified by the methods of the present invention and a carrier, such as a pharmaceutically acceptable carrier. A "pharmaceutically acceptable carrier" includes pharmaceutically acceptable excipients and/or pharmaceutically acceptable delivery vehicles, which are suitable for use in administration of the composition to a suitable in vitro, ex vivo or in vivo site. A suitable in vitro, in vivo or ex vivo site is preferably at or near a cell that expresses a protein serine/threonine phosphatase, and most preferably, at or near a site of interest in the patient. In one embodiment, the pharmaceutically acceptable carrier is capable of maintaining a compound identified by the present methods in a form that, upon arrival of the compound at the cell target in a culture or in patient, the compound is capable of interacting with its target (e.g., the catalytic subunit of the protein serine/threonine phosphatase).

Suitable excipients of the present invention include excipients or formularies that transport or help transport, but do not specifically target a composition to a cell (also referred to herein as non-targeting carriers). Examples of pharmaceutically acceptable excipients include, but are not limited to water, phosphate buffered saline, Ringer's solution, dextrose solution, serum-containing solutions, Hank's solution, other aqueous physiologically balanced solutions, oils, esters and glycols. Aqueous carriers can contain suitable auxiliary substances required to approximate the physiological conditions of the recipient, for example, by enhancing chemical stability and isotonicity.

Suitable auxiliary substances include, for example, sodium acetate, sodium chloride, sodium lactate, potassium chloride, calcium chloride, and other substances used to produce phosphate buffer, Tris buffer, and bicarbonate buffer. Auxiliary substances can also include preservatives, such as thimerosal, formalin and benzol alcohol. Compositions of the present invention can be sterilized by conventional methods and/or lyophilized.

One type of pharmaceutically acceptable carrier includes a controlled release formulation that is capable of slowly releasing a composition of the present invention into a patient or culture. As used herein, a controlled release formulation comprises a compound of the present invention {e.g., a protein (including homologues), a drug, an antibody, a nucleic acid molecule, or a mimetic) in a controlled release vehicle.

Suitable controlled release vehicles include, but are not limited to, biocompatible polymers, other polymeric matrices, capsules, microcapsules, microparticles, bolus preparations, osmotic pumps, diffusion devices, liposomes, lipospheres, and transdermal delivery systems. Other carriers of the present invention include liquids that, upon administration to a patient, form a solid or a gel in situ. Preferred carriers are also biodegradable (i.e., bioerodible). When the compound is a recombinant nucleic acid molecule, suitable delivery vehicles include, but are not limited to liposomes, viral vectors or other delivery vehicles, including ribozymes. Natural lipid- containing delivery vehicles include cells and cellular membranes. Artificial lipid- containing delivery vehicles include liposomes and micelles. A delivery vehicle of the present invention can be modified to target to a particular site in a patient, thereby targeting and making use of a compound of the present invention at that site. Suitable modifications include manipulating the chemical formula of the lipid portion of the delivery vehicle and/or introducing into the vehicle a targeting agent capable of specifically targeting a delivery vehicle to a preferred site, for example, a preferred cell type. Other suitable delivery vehicles include gold particles, poly-L-lysine/DNA- molecular conjugates, and artificial chromosomes.

A pharmaceutically acceptable carrier which is capable of targeting is herein referred to as a "delivery vehicle." Delivery vehicles are capable of delivering a composition of the present invention to a target site in a patient. A "target site" refers to a site in a patient to which one desires to deliver a composition. For example, a target site can be any cell which is targeted by direct injection or delivery using liposomes, viral vectors or other delivery vehicles, including ribozymes and antibodies. Examples of delivery vehicles include, but are not limited to, artificial and natural lipid-containing delivery vehicles, viral vectors, and ribozymes. Natural lipid-containing delivery vehicles include cells and cellular membranes. Artificial lipid-containing delivery vehicles include liposomes and micelles. A delivery vehicle of the present invention can be modified to target to a particular site in a subject, thereby targeting and making use of a compound of the present invention at that site. Suitable modifications include manipulating the chemical formula of the lipid portion of the delivery vehicle and/or introducing into the vehicle a compound capable of specifically targeting a delivery vehicle to a preferred site, for example, a preferred cell type. Specifically, targeting refers to causing a delivery vehicle to bind to a particular cell by the interaction of the compound in the vehicle to a molecule on the surface of the cell. Suitable targeting compounds include ligands capable of selectively (i.e., specifically) binding another molecule at a particular site. Examples of such ligands include antibodies, antigens, receptors and receptor ligands. Manipulating the chemical formula of the lipid portion of the delivery vehicle can modulate the extracellular or intracellular targeting of the delivery vehicle. For example, a chemical can be added to the lipid formula of a liposome that alters the charge of the lipid bilayer of the liposome so that the liposome fuses with particular cells having particular charge characteristics. In one embodiment, a targeting carrier can be a portion of a protein serine/threonine phosphatase as described elsewhere herein, which is linked to the compound. A composition which includes a compound identified according to the present methods can be delivered to a cell culture or patient by any suitable method. Selection of such a method will vary with the type of compound being administered or delivered {i.e., protein, peptide, nucleic acid molecule, mimetic, or other type of compound), the mode of delivery {i.e., in vitro, in vivo, ex vivo) and the goal to be achieved by administration/delivery of the compound or composition. According to the present invention, an effective administration protocol {i.e., administering a composition in an effective manner) comprises suitable dose parameters and modes of administration that result in delivery of a composition to a desired site {i.e., to a desired cell) and/or in the desired regulatory event {e.g., to potentiate protein serine/threonine phosphatase activity).

Administration routes include in vivo, in vitro and ex vivo routes. In vivo routes include, but are not limited to, oral, nasal, intratracheal injection, inhaled, transdermal, rectal, and parenteral routes. Preferred parenteral routes can include, but are not limited to, subcutaneous, intradermal, intravenous, intramuscular and intraperitoneal routes. Intravenous, intraperitoneal, intradermal, subcutaneous and intramuscular administrations can be performed using methods standard in the art. Aerosol (inhalation) delivery can also be performed using methods standard in the art (see, for example, Stribling et al., PNAS 189:1 1277-1 1281 , 1992, which is incorporated herein by reference in its entirety). Oral delivery can be performed by complexing a therapeutic composition of the present invention to a carrier capable of withstanding degradation by digestive enzymes in the gut of a patient. Examples of such carriers include plastic capsules or tablets, such as those known in the art. Direct injection techniques are particularly useful for suppressing graft rejection by, for example, injecting the composition into the transplanted tissue, or for site-specific administration of a compound, such as at the site of a tumour.

It will be apparent to one of skill in the art that the number of doses of a compound identified by the methods of the present invention (as herein described) as capable of modulating protein serine/threonine phosphatase activity administered to a patient in need thereof will depend on the severity of the condition and the response of an individual to the treatment. It is within the scope of the present invention that a suitable number of doses, as well as the time periods between administrations, includes any number required to cause regression of the condition. As discussed infra, it is well within the ability of a person of ordinary skill to ascertain a therapeutically effective dose. For example, the dosage may be arrived at by theoretical means only, and may be based on body weight, age, height, or surface area of the subject. Alternatively, the dosage may be arrived at empirically, for example by titrating from a low dosage to a higher dosage. While titrating, the subject is assessed clinically for a favourable response to the treatment, as well as for side effects. Once an acceptable response is achieved (with an acceptable side effect profile), the dosage is maintained at that level for the remainder of the treatment period. A therapeutically effective dosage may be 3 μg/kg to 20 mg/kg per day, 0.015 mg/kg to 20 mg/kg, 0.15 mg/kg to 20.0 mg/kg, 0.1 mg/kg to 14 mg/kg, 0.1 mg/kg to 13 mg/kg, 0.1 mg/kg to 12 mg/kg, 0.1 mg/kg to 10 mg/kg, 0.1 mg/kg to 9 mg/kg, 0.1 mg/kg to 8 mg/kg, 0.1 mg/kg to 7 mg/kg, 0.1 mg/kg to 6 mg/kg, 0.15 mg/kg to 5 mg/kg, 0.15 mg/kg to 4 mg/kg, 0.15 mg/kg to 3 mg/kg, 0.15 mg/kg to 2 mg/kg, 0.15 mg/kg to 1 mg/kg, especially 0.07 mg/kg to 6.5 mg/kg or 0.1 mg/kg to 14 mg/kg or 0.15 mg/kg to 5 mg/kg per day, and more especially 0.07 mg/kg to 2 mg/kg per day.

In certain embodiments of the methods of treatment, the composition is administered in combination with at least one cytostatic agent or cytotoxic agent. Non- limiting examples of cytostatic agents are selected from: (1 ) kinase inhibitors, illustrative examples of which include Iressa®, Gleevec, Tarceva™, (Erlotinib HCI), BAY-43- 9006, inhibitors of the split kinase domain receptor tyrosine kinase subgroup (e.g., PTK787/ZK 222584 and SUI 1248); (2) receptor kinase targeted antibodies, which include, but are not limited to, Trastuzumab (Herceptin®), Cetuximab (Erbitux®), Bevacizumab (Avastin™), Rituximab (ritusan®), Pertuzumab (Omnitarg™); (3) mTOR pathway inhibitors, illustrative examples of which include rapamycin and CCI-778; (4) Apo2L/Trail, anti-angiogenic agents such as but not limited to endostatin, combrestatin, angiostatin, thrombospondin and vascular endothelial growth inhibitor (VEGI); (5) antineoplastic immunotherapy vaccines, representative examples of which include activated T-cells, non-specific immune boosting agents (i.e., interferons, interleukins); (6) hormonal antineoplastic agents, non-limiting examples of which include Nilutamide, Cyproterone acetate, Anastrozole, Exemestane, Tamoxifen, Raloxifene, Bicalutamide, Aminoglutethimide, Leuprorelin acetate, Toremifene citrate, Letrozole, Flutamide, Megestrol acetate and Goserelin acetate; (7) gonadal hormones such as but not limited to Cyproterone acetate and Medoxyprogesterone acetate; (8) antimetabolites, illustrative examples of which include Cytarabine, Fluorouracil, Gemcitabine, Topotecan, Hydroxyurea, Thioguanine, Methotrexate, Colaspase, Raltitrexed and Capicitabine; (9) anabolic agents, such as but not limited to, Nandrolone; (10) adrenal steroid hormones, illustrative examples of which include Methylprednisolone acetate, Dexamethasone, Hydrocortisone, Prednisolone and Prednisone.

In some embodiments, the cytostatic agent is a nucleic acid molecule, suitably an antisense or siRNA recombinant nucleic acid molecule. In other embodiments, the cytostatic agent is a peptide or polypeptide. In still other embodiments, the cytostatic agent is small molecule. The cytostatic agent may be a cytotoxic agent that is suitably modified to enhance uptake or delivery of the agent. Non-limiting examples of such modified cytotoxic agents include, but are not limited to, pegylated or albumin-labelled cytotoxic drugs.

The compositions of the present invention may be employed in combination with other known treatments for tumors, for instance but not limited to, surgery, chemotherapy and radiotherapy. In some embodiments, the selenate or its pharmaceutically acceptable salt is used in combination with radiotherapies, such as but not limited to, conformal external beam radiotherapy (50-100 Grey given as fractions over 4-8 weeks), either single shot or fractionated, high dose rate brachytherapy, permanent interstitial brachytherapy, systemic radio-isotopes (e.g., Strontium 89). In some embodiments the radiotherapy may be administered in combination with a radiosensitizing agent. Illustrative examples of radiosensitizing agents include but are not limited to efaproxiral, etanidazole, fluosol, misonidazole, nimorazole, temoporfin and tirapazamine. In other embodiments, selenate or its pharmaceutically acceptable salt is used in combination with a tumorectomy.

In some embodiments the composition may be administered in combination with 5- alpha reductase inhibitors such as finasteride or dutasteride; alpha adrenergic receptor blockers such as terazosin (Hytrin), doxazosin (Cardura), alfuzosin, tamsulosin (flomax) and Prazosin (Minipress); selective estrogen receptor modulators

(SERMs) such as toremifene citrate (Acapodene), tamoxifen and raloxifen; selective androgen receptor modulators (SARMs) such as prostarine; or phytotherapeutics such as those derived from the American dwarf palm tree (saw palmetto or Serenoa repens), African plum tree (Pygeum africanum), pumpkin seeds, rye pollen extracts, South African star grass roots, stinging nettle roots and the purple cone flower.

In some embodiments, the composition may be administered in combination with non steroidal anti inflammatory drugs such as aspirin, ibuprofen, naproxene; or Cox-2 inhibitors such as sulindac, celocoxib (celebrex), refocoxib (vioxx) and lumiracoxib.

In some embodiments the composition may be administered in combination with selective estrogen receptor modulators such as tamoxifen, raloxifen and toremifene citrate.

In some embodiments the composition may be administered in combination with progesterone receptor antagonists such as mifepristone; immune modulating agents such as interferon; chemotherapy such as hydroxyurea and external beam radiation.

In certain embodiments of the methods of treatment, the composition is administered in combination with a hormone ablation therapy and at least one cytostatic agent or cytotoxic agent. Non-limiting examples of cytostatic agents are selected from: (1 ) microtubule-stabilizing agents such as but not limited to taxanes, paclitaxel, docetaxel, epothilones and laulimalides; (2) kinase inhibitors, illustrative examples of which include Iressa®, Gleevec, Tarceva™, (Erlotinib HCI), BAY-43-9006, inhibitors of the split kinase domain receptor tyrosine kinase subgroup (e.g., PTK787/ZK 222584 and SUI 1248); (3) receptor kinase targeted antibodies, which include, but are not limited to, Trastuzumab (Herceptin®), Cetuximab (Erbitux®), Bevacizumab (Avastin™), Rituximab (ritusan®), Pertuzumab (Omnitarg™); (4) mTOR pathway inhibitors, illustrative examples of which include rapamycin and CCI-778; (5) Apo2L/Trail, anti- angiogenic agents such as but not limited to endostatin, combrestatin, angiostatin, thrombospondin and vascular endothelial growth inhibitor (VEGI); (6) antineoplastic immunotherapy vaccines, representative examples of which include activated T-cells, non-specific immune boosting agents (i.e., interferons, interleukins); (7) antibiotic cytotoxic agents such as but not limited to doxorubicin, bleomycin, dactinomycin, daunorubicin, epirubicin, mitomycin and mitozantrone; (8) alkylating agents, illustrative examples of which include Melphalan, Carmustine, Lomustine, Cyclophosphamide, Ifosfamide, Chlorambucil, Fotemustine, Busulfan, Temozolomide and Thiotepa; (9) hormonal antineoplastic agents, non-limiting examples of which include Nilutamide, Cyproterone acetate, Anastrozole, Exemestane, Tamoxifen, Raloxifene, Bicalutamide, Aminoglutethimide, Leuprorelin acetate, Toremifene citrate, Letrozole, Flutamide, Megestrol acetate and Goserelin acetate; (10) gonadal hormones such as but not limited to Cyproterone acetate and Medoxyprogesterone acetate; (1 1 ) antimetabolites, illustrative examples of which include Cytarabine, Fluorouracil, Gemcitabine, Topotecan, Hydroxyurea, Thioguanine, Methotrexate, Colaspase, Raltitrexed and Capicitabine; (12) anabolic agents, such as but not limited to, Nandrolone; (13) adrenal steroid hormones, illustrative examples of which include Methylprednisolone acetate, Dexamethasone, Hydrocortisone, Prednisolone and Prednisone; (14) neoplastic agents such as but not limited to Mnotecan, Carboplatin, Cisplatin, Oxaliplatin, Etoposide and Dacarbazine; and (15) topoisomerase inhibitors, illustrative examples of which include topotecan and irinotecan.

In specific embodiments, the cytostatic agent is a microtubule stabilizing agent, especially a taxane and preferably paclitaxel. In some embodiments, the cytotoxic agent is selected from the anthracyclines such as idarubicin, doxorubicin, epirubicin, daunorubicin and mitozantrone, CMF agents such as cyclophosphamide, methotrexate and 5-fluorouracil or other cytotoxic agents such as cisplatin, carboplatin, bleomycin, topotecan, irinotecan, melphalan, chlorambucil, vincristine, vinblastine and mitomycin-C. Therapeutically effective amounts of cytostatic agents and cytotoxic agents may be those that would normally be used in the absence of the composition. Alternatively, the therapeutically effective amount of the cytotoxic agent or cytostatic agent is lower than used in the absence of the combination of the composition and hormone ablation therapy.

In preferred embodiments the composition is administered in conjunction with hormone ablation therapy such as surgical castration, fϊnesteride, Nilutamide, Cyproterone acetate, Bicolutamide, Leuprorelin acetate, Flutamide and Goserelin acetate. In other preferred embodiments, the hormone-dependent cancer is breast cancer and the hormone ablation therapy is selected from Anastrozole; Exemestane, Tamoxifen, Aminoglutethimide, Toremifene citrate, Letrozole, Megestrol acetate and Goserelin acetate. In still other embodiments, the composition is administered together with a hormone ablation therapy such as progestins such as megestrol acetate, levinorgestrol and norgestrol. In some embodiments, the therapy further comprises a cytostatic agent, particularly a microtubule stabilizing agent, especially a taxane, more especially paclitaxel. In a further aspect, the present invention provides use of a compound as described herein in the manufacture of a medicament for the treatment or prevention of a condition associated with the activity of a serine/threonine protein phosphatase.

Methods of Identifying Modulators of Protein Serine/Threonine Phosphatase Activity In one aspect of the present invention, there is provided a method of identifying a compound capable of modulating protein serine/threonine phosphatase activity, the method comprising: (a) contacting the protein serine/threonine phosphatase, or an analogue or a fragment thereof, with a candidate compound under conditions permitting binding of the test compound to the protein serine/threonine phosphatase; and (b) determining whether the test compound binds to one or more binding motifs on the catalytic subunit of the protein serine/threonine phosphatase or to an analogue or fragment thereof.

As used herein the term "identifying" encompasses either designing a new compound, selecting a compound from a group or library of previously known compounds or modifying a selected compound.

Protein serine/threonine phosphatases are divided into two families namely: phospho protein phosphatase (PPP) and protein phosphatase magnesium dependent (PPM), according to amino acid sequence homology, protein structure, and sensitivity to inhibitors. There are several isoforms of protein serine/threonine phosphatases, comprising PP1 , PP2A, PP2B, PP2C, PP4, PP5, PP6 and PP7, as well as their various isoforms. Even within the same PPP family significant structural diversity is present. Studies have shown that the catalytic domains of these phosphatases have a high degree of identity. However, the ability of these enzymes to form heteromeric complexes with a variety of regulatory subunits makes them unique. These regulatory domains or subunits localize the protein complexes to a specific subcellular compartment, modulate the substrate specificity, or alter catalytic activity.

In one embodiment of the present invention, the protein serine/threonine phosphatase is selected from the group consisting of PP1 α, PP1 β, PP1γ1 , PP1γ2, PP2A, PP2B (calcineurin), PP2C, PP4, PP5 and PP6. In a further embodiment, the protein serine/threonine phosphatase according to the present invention is PP2A or PP5. Analogues and Fragments

As used herein, the term "fragment typically refers to a fragment of a protein serine/threonine phosphatase, or an analogue thereof, that retains its ability to bind a candidate compound, such as a candidate compound that is capable of modulating protein serine/threonine phosphatase activity. Such fragments may comprise at least 5 amino acid residues, more preferably at least 10 amino acid residues, and still more preferably at least 20 amino acid residues of the native enzyme. Such fragments may include the scaffold, regulatory and/or catalytic subunits of a protein serine/threonine phosphatase, or fragments thereof.

Fragments of interest include, but are not limited to, fragments of at least about 20 contiguous amino acids, more usually at least about 50 contiguous amino acids, and may comprise 100 or more amino acids, up to the complete protein, and may extend further to include additional sequences. It is expected that a fragment of the protein serine/threonine phosphatase retain at least one binding motif, as herein described. The term "binding motif, as used herein, generally refers to a region of a molecule or molecular complex that, as a result of its shape, charge, hydrophobicity or hydrophilicity, is able to interact with another chemical entity. Thus, a binding motif may include or consist of features such as cavities, pockets, surfaces, or interfaces between domains. Chemical entities that may interact with a binding domain include, but are not limited to, cofactors, substrates, inhibitors, agonists, and antagonists.

As used herein, the term "analogue" typically denotes a protein serine/threonine phosphatase, or fragment thereof, that has an amino acid sequence that is substantially identical to the amino acid sequence of the naturally occurring protein serine/threonine phosphatase.

The term "substantially identical', as used in regards to an analogue, typically denotes a substitution or addition of one or more amino acids such that the resulting analogue has at least some of the biological activity of the naturally occurring enzyme. Analogues may be naturally occurring, such as an allelic variant or an mRNA splice variant, or they may be constructed using synthetic or recombinant techniques available to one skilled in the art.

As used herein, the term "variant' typically denotes an enzyme, or a fragment thereof, that exhibits an amino acid sequence that is at least 80% identical to the native enzyme. Also contemplated are embodiments in which a variant comprises an amino acid sequence that is at least 90% identical, optionally at least 95% identical, optionally at least 98% identical, optionally at least 99% identical, or optionally at least 99.9% identical to the native molecule. Percent identity may be determined by visual inspection and/or mathematical calculation by methods known to those skilled in the art. Variants may be naturally occurring, synthetic or recombinant.

In one embodiment of the present invention, a variant of a protein serine/threonine phosphatase, or a fragment thereof, includes an enzyme or fragment thereof that is substantially homologous to the native form of the enzyme, but which has an amino acid sequence different from that of the native form because of one or more deletions, insertions or substitutions. Certain embodiments include amino acids that comprise from one to ten deletions, insertions or substitutions of amino acid residues when compared to a native sequence. A given sequence may be replaced, for example, by a residue having similar physiochemical characteristics. Examples of such conservative substitution of one aliphatic residue for another, such as lie, VaI, Leu or Ala for one another; substitution of one polar residue for another, such as between Lys and Arg,or GIu and Asp, or GIn and Asn; or substitutions of one aromatic residue for another, such as Phe, Trp or Tyr for one another. Other conservative substitutions, e.g., involving substitutions of entire regions having similar hydrophobicity characteristics, are well known in the art. Variants may also be generated by the truncation of a native enzyme sequence.

A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains {e.g., lysine, arginine, histidine), acidic side chains {e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains {e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains {e.g., threonine, valine, isoleucine) and aromatic side chains {e.g., tyrosine, phenylalanine, tryptophan). Thus, an amino acid residue of a protein serine/threonine phosphatase is preferably replaced with another amino acid residue from the same side chain family. In a preferred embodiment, mutations can be introduced randomly along all or part of the enzyme coding sequence, such as by saturation mutagenesis. The resultant mutants can be screened to identify variants that demonstrate at least some of the biological activity of the native enzyme. Following mutagenesis, the encoded protein can be expressed recombinantly and the activity of the enzyme can be determined by the methods described herein.

The protein serine/threonine phosphatase of the present invention, or an analogue or a fragment thereof, may be prepared by in vitro synthesis using conventional methods as known in the art. Various commercial synthetic apparatuses are available, for example, automated synthesizers {e.g., CS936X Peptide Synthesizer, CSBio Company, Inc.). Using such synthesizers, a skilled person can readily substitute for the naturally occurring amino acids one or more unnatural amino acids. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like. If desired, various groups can be introduced into the protein during synthesis that allow for linking to other molecules or to a surface. For example, cysteines can be used to make thioethers, histidines can be used for linking to a metal ion complex, carboxyl groups can be used for forming amides or esters, amino groups can be used for forming amides, and the like.

Identification of Binding Motifs Determination of the three-dimensional structure of the catalytic subunit of a protein serine/threonine phosphatase either alone or in complex with a ligand by X-ray crystallography provides high-resolution and very high quality information about the molecular recognition of the compound in the catalytic subunit. The applicants have performed rationalized candidate compound optimization studies through computer based modelling of sulphate and selenate in complex with the protein serine/threonine phosphatase PP2A, and analysed their molecular interactions and identified a number of binding motifs implicated in the modulation of protein serine/threonine phosphatase activity.

In one embodiment of the present invention, the binding motifs include one or more amino acid residues selected from the group of amino acid clusters consisting of:

(a) D57, H59, D85, R89, N117, H118, H167, R214, H241;

(b) D57, H59, D85, N117, H167, H241;

(C) 162,163,235,236,250,254,278,282,284; (d) N1 17,H1 18,S120,R121 ,Q122,l123,T124,E188,V189,P190,H191 ,G192,C196, W200, A216,G217 (e) I14,N18,M66,F69,R7O,G73,K74,S75,Y8O,L99,L1O3;

(f) F164,L166,G169,L17O,S171,I174,I18O,L198,W2O9,F228;

(g) L39,Y86,V97,V101,l113,E119,F146,L149,F150,L153; (h) Y86,T96,V97,L100,V101,l113,F150,L153; (i) Q46,E47,V48,R49,P51,V52,N79,Y80,L81,K104,R108,E109,R110,1111.T112;

C) L31,A35,L39,L100,V101,K104,V105,l113,L149,L153;

(k) D57,V58,Q61 ,D64,L65,L68,S261 ,A262,P263,F289;

(I) C55,G56,D57,V58,H59,L81,F82,M83,G84,D85,Y86,L100,L114,S261; (m) D57,H59,D85,R89,N117,H118,l123,Y127,W200,R214,H241,F260,Y265; (n) V28,K29,C32,E33,K36,K144,Y145,D148,L149;

(o) I14,S75,P76,L1O3,R1O6,Y1O7,I111;

(P) L39,T40,D151,Y152,L153,R185,L186,Q187;

(q) H191,E192,G193,C196,D197,A216,G217,Y218;

(r) Y107,R108,E109,R110; (s) P51,V52,T53,D77,T78,N79,Y80,M276,E277,L278,D279 ;

(t) D131,L134,R135,Y307,F308;

(u) V244,M245,E246,G247,Y248,N249,W250;

(v) W200,S201,D202,S212,P213,R214,A216,G217,Y218,T219,F220;

(w) R89,R214,H241,Q242,L243,F260,Y265; (X) F6,W13,S30,L31,E33,K34,E37;

(y) L10,D11,l14,E15,P76,R106,Y107;

(Z) G90,Y91,Y92,S93,G128,F129,D131,E132,R135,K136;

(aa) L17O,S171,I174,D175,W2O9,I224,T227,F228;

(bb) H59,D85,D88,R89,H118,Y127,Y265; (CC) Y92,Y267,K294,R295,G296,E297,P298,V308;

(dd) P203,D204,D205,R206,G207,G221,Q222,D223,Q242,N249,C251,H252,D253;

(ee) L183,D184,R185,Q187,P190,P194,H195;

(ff) Y267,P291,A292,R294;

(gg) P203, R239,Q242, L243,V244, N249.T258; (hh) E67,l71,F289,D290,P291,A292,P293;

(ii) E33,K36,E37;

Cj) V5,F6,T7,K8,E9;

(kk) I211,S212,G215,A216,G217,Y218;

(II) E226,N229,H230,L234,N255; (mm) T176,L177,D178,R181,N232;

(nn) H63,H66,E67,R70,A292,P293,R294; (oo) H63,Y92,Y267,R294,R295;

(PP) D202,P203,D204,P213.T219,F220,Q242;

(qq) V300,R303,T304,P305;

(rr) H63,P293,R294,R295; (SS) T40,N44,L183,D184,R185,L186;

(tt) E42,Q46,V48,K104,R108,E109,l111,T112;

(UU) D204,R206,G210,l21 1 ,S212,P213,T219;

(W) D204,R206,l211 ,P213,T219; and

(WW) N18,E19,C20,F62,H62,M63. (XX) R89, N117, H1 18, 1123, Y127, W200, P213, R214, H241 , Q242, L243, F260,

Y265;

(yy) N1 17, S120, R121 , Q122, 1123, Y127, E188, V189, P190, H191 , W200;

(ZZ) D204, D205, C221 , Q222, G251 , H252, D253;

(aaa) R89, R214; (bbb) H191. Q122;

(CCC) E188, R121 ;

(ddd) H252, D253; and

(eee) D290.

The skilled person understands that the above-referenced residues may not bind directly to any compound, and an intermediate atom, ion or molecule may be involved. For example, in one form of the invention a Mn2+ ion is involved in binding, with the ion being interdisposed between the residue and the compound. Alternatively, or additionally, a water molecule may be involved in the binding. In one embodiment, a water and/or Mn2+ ion is involved in the binding of a compound to a residue selected from the group consisiting of D57, H59, D85, H167, N1 17, H241 R214, and Y267. According, in one form of the method, the binding motif is a component of a binding complex, the binding complex comprising an accessory atom, ion or molecule.

In another embodiment of the present invention, the method includes isolating the test compound that has been identified as capable of modulating protein serine/threonine phosphatase activity.

As used herein, the terms "isolating', "isolated' and the like do not reflect the extent to which the protein has been purified. In one embodiment, an isolated compound is produced recombinantly. In another embodiment, the isolated compound may be purified from any number of natural sources by methods familiar to those skilled in the art. The isolated compound may therefore include purified, partially purified, recombinant, mutated/modified and synthetic proteins.

Proteins of the present invention may be retrieved, obtained, and/or used in "substantially pure" form. As used herein, "substantially pure" generally refers to a purity that allows for the effective use of the compound in vitro, ex vivo or in vivo according to the present invention. For a protein to be useful in an in vitro, ex vivo or in vivo method according to the present invention, it is substantially free of contaminants, other proteins and/or chemicals that might interfere or that would interfere with its use in a method disclosed by the present invention, or that at least would be undesirable for inclusion with the candidate compound when it is used in a method according to an aspect of the present invention. In one embodiment, a "substantially pure" compound, as referenced herein, is a protein that can be produced by any method (i.e., by direct purification from a natural source, recombinantly, or synthetically), and that has been purified from other components such that the compound comprises at least about 80% weight/weight of the total protein in a given composition (e.g., the protein is about 80% of the composition in a solution/composition/buffer), at least about 85%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% weight/weight of the total protein in a given composition.

The step of identifying a compound in the method of the present invention can include any suitable method of drug design, drug screening or identification, comprising: directed drug design, random drug design, grid-based drug design, and/or computational screening of one or more databases of chemical compounds.

In Silico Screening Methods Information provided by crystallising an enzyme and elucidating the structural relationship of one or more of the binding motifs can be used in computer aided drug design applications. X-ray structure coordinates obtained when a crystal structure is solved define a unique configuration of three-dimensional points that serve to identify positions of the atoms in the crystal relative to each other. The data obtained does not, however, define an absolute set of points in space. As would be understood by a skilled addressee a set of structure coordinates for any crystal structure defines a relative set of points that, in turn, define a configuration of atoms in three dimensions. In essence the importance of crystal structure data is that it provides information as to the spatial relationship of the atoms in the crystal with respect to each other. It would be understood that a similar or identical configuration could just as easily be defined by an entirely different set of coordinates, provided the distances and angles between coordinates remained essentially the same. In addition, a scalable configuration of points can be defined by increasing or decreasing the distances between coordinates by a scalar factor while keeping the angles essentially the same. It is the relative information provided by the data set that is important in determining its utility for drug design applications not the absolute value of any of the data points obtained. An exemplary crystal structure is shown in the figures and represented by the data shown in Figures 13. When using this data in rational drug design, only a portion of the structural coordinates are in fact needed as the important information is that detailing the relative conformation of the atoms constituting the binding site and/or point of interaction of the two molecules in the complex as the case may be. Thus a person could exploit the advance made by the inventors by only utilizing a portion of the data and not the entire data set. This would be sufficient to allow a skilled user to then use computer aided drug design programs. Whilst the best drug design/modelling would be carried out using all the crystal data pertaining to the binding motifs identified by the inventors, it is possible to use only a portion of the binding motif data and still get workable material. It is theoretically possible, therefore, that useful information could be obtained merely by including data from 2 points within the domain although the more points that are included the better the modelling will become.

The present invention therefore includes within its scope data sets derived from the crystal structure data coordinates representing one or more of the binding motifs on the catalytic subunit of a protein serine/threonine phosphatase as herein described or data sets defining the same relative spatial configuration of atoms as the structural coordinates of the crystal structure data provided by the present invention, as herein described. As discussed above, in most applications it is envisaged that only a portion of the data set will be needed. The invention therefore includes data sets where only a portion of the data set of the present invention is utilized. In one embodiment, the data set includes data for at least 3 atoms derived from or defining the same relative spatial configuration as the data of the present invention. In a more preferred embodiment the data set includes 5 atoms, even more preferably 7 atoms, more preferably 1 1 atoms, even more preferably 20 atoms either derived from or defining the same relative structural configuration as the structural coordinates for atoms given in RCSB Protein Databank under accession 2iae

(http://www.rcsb.org/pdb/files/2iae.pdb); as accessed 7 December 2007.

Thus, in a second aspect of the present invention, there is provided a method of identifying a compound which is capable of binding to a protein serine/threonine phosphatase, the method comprising: (a) providing a three dimensional structure of a catalytic subunit of a protein serine/threonine phosphatase, or an analogue or fragment thereof; and (b) identifying a candidate compound for binding to one or more binding motifs on the catalytic subunit, or the analogue or fragment thereof by performing structure-based drug design with the structure of (a).

In one embodiment of the present invention, the three dimensional structure of the catalytic subunit of the protein serine/threonine phosphatase is provided by using the atomic coordinates described supra.

In another aspect of the present invention there is provided a method of identifying a candidate compound capable of binding to a protein serine/threonine phosphatase, the method comprising: (a) identifying a candidate compound that has a conformation and polarity such that it interacts with at least one relevant amino acid residue of one or more binding motifs on a catalytic subunit of the protein serine/threonine phosphatase, or an analogue or fragment thereof.

In one embodiment, the method of the present invention includes the steps of: (a) providing a three dimensional structure of a catalytic subunit of a protein serine/threonine phosphatase as herein described; (b) identifying a candidate compound for binding to the protein serine/threonine phosphatase by performing structure based drug design with the structure of (a) to identify a candidate compound that binds to one or more binding motifs on the catalytic subunit of the protein serine/threonine phosphatase; (c) contacting the candidate compound identified in step (b) with a cell that expresses the protein serine/threonine phosphatase under conditions in which the candidate compound can bind to the protein serine/threonine phosphatase; and (d) measuring the activity of the protein serine/threonine phosphatase before and after contact with the candidate compound, wherein a candidate compound is selected as a compound that modulates the activity of the protein serine/threonine phosphatase in the cell, as compared to in the absence of the candidate compound.

The structures used to perform the above-described method have been described in detail above and in the Figures. According to the present invention, the phrase "providing a three dimensional structure of a catalytic subunit of a protein serine/threonine phosphatase" is defined as any means of providing, supplying, accessing, displaying, retrieving, or otherwise making available the three dimensional structure of the catalytic subunit of a protein serine/threonine phosphatase as herein described herein. For example, the step of providing can include, but is not limited to, accessing the atomic coordinates for the structure from a database; importing the atomic coordinates for the structure into a computer or other database; displaying the atomic coordinates and/or a model of the structure in any manner, such as on a computer, on paper, etc. and determining the three dimensional structure of the catalytic subunit of a protein serine/threonine phosphatase de novo using the guidance provided herein.

As used herein, a "structure" of a protein refers to the components and the manner of arrangement of the components to constitute the protein. The "three dimensional structure" or "tertiary structure" of the protein refers to the arrangement of the components of the protein in three dimensions. Such term is well known to those of skill in the art. It is also to be noted that the terms "tertiary and "three dimensional' can be used interchangeably.

The second step of the method of structure based identification of compounds of the present invention includes identifying a candidate compound for binding to one or more binding motifs on the catalytic subunit, or the analogue or fragment thereof by performing structure-based drug design with the structure of (a). The inventors have shown that selenate can bind to the catalytic subunit of a protein serine/threonine phosphatase and has been shown to modulate (i.e., enhance) the activity of a protein serine/threonine phosphatases such as PP2A. Therefore, identification and/or design of compounds that mimic, enhance, disrupt or compete with the interactions of selenate with the catalytic subunit of a protein serine/threonine phosphatase are also desirable and thus form another aspect of the present invention. In another aspect of the present invention there is provided a method of identifying a candidate compound capable of binding to a protein serine/threonine phosphatase, the method comprising: (a) identifying a candidate compound that has a conformation and polarity such that it interacts with at least one relevant amino acid residue of one or more binding motifs on a catalytic subunit of the protein serine/threonine phosphatase, or an analogue or fragment thereof.

In another aspect of the present invention there is provided a computer-assisted method for identifying a candidate compound capable of binding to a protein serine/threonine phosphatase, the method comprising: (a) supplying a computer modelling application with a set of structure coordinates of a molecule or molecular complex, at least a portion of the structural coordinates of the molecule or molecular complex being derived from, or defining the same relative spatial configuration as, at least a portion of the atomic coordinates of one or more binding motifs on a catalytic subunit of the protein serine/threonine phosphatase;.. (b) supplying the computer modelling application with a set of structure coordinates of the candidate compound; and (c) determining whether the candidate compound is expected to bind to the molecule or molecular complex, wherein binding to the molecule or molecular complex is indicative of potential binding to the catalytic subunit of the protein serine/threonine phosphatase.

In another aspect of the present invention there is provided a computer-assisted method for designing a candidate compound capable of binding to a catalytic subunit of a protein serine/threonine phosphatase, the method comprising: (a) supplying a computer modelling application with a set of structure coordinates of a molecule or molecular complex, at least a portion of the structural coordinates of the molecule or molecular complex being derived from, or defining the same relative spatial configuration as, at least a portion of the atomic coordinates of one or more of the binding motifs of the catalytic subunit of the protein serine/threonine phosphatase; (b) supplying the computer modelling application with a set of structure coordinates for the candidate compound; (c) evaluating the potential binding interactions between the candidate compound and substrate binding pocket of the molecule or molecular complex; (d) structurally modifying the candidate compound to yield a set of structure coordinates for a modified candidate compound; and (e) determining whether the modified candidate compound is expected to bind to the molecule or molecular complex, wherein binding to the molecule or molecular complex is indicative of potential binding to the catalytic subunit of the protein serine/threonine phosphatase.

In another aspect of the present invention there is provided a computer-assisted method for designing a candidate compound capable of binding to a catalytic subunit of a protein serine/threonine phosphatase de novo, the method comprising: (a) supplying a computer modelling application with a set of structure coordinates of a molecule or molecular complex, at least a portion of the structural coordinates of the molecule or molecular complex being derived from, or defining the same relative spatial configuration as, at least a portion of the atomic coordinates of one or more of the binding motifs of the catalytic subunit of the protein serine/threonine phosphatase; (b) computationally building a candidate compound represented by a set of structure coordinates; and (c) determining whether the candidate compound is expected to bind to the molecule or molecular complex, wherein binding to the molecule or molecular complex is indicative of potential binding to the catalytic subunit of the protein serine/threonine phosphatase.

Traditionally drug development and design has occurred by pharmaceutical companies undertaking large screening programs, by the use of structure activity studies or by serendipity. Whilst these techniques are successful to a point they are labour intensive and rely on luck and the breadth of the study rather than rigorous principles of the desired interaction at a molecular level. More recently drug design has been carried out by using computer assisted models which can model the desired interaction hopefully leading to improved therapeutic compounds. Computers therefore provide the drug developer with the ability to screen, identify, select and/or design chemical entities capable of associating the particular receptor of interest. As would be clear, however, before this can be carried out detailed knowledge of the structure of the receptor must be known so that this data can be input into the computer software that carries out the modelling. Detailed knowledge of the exact structural coordinates of a receptor permits the design and/or identification of synthetic compounds and/or other molecules which are spatially adapted for optimal interaction with the binding site of the receptor. Accordingly computer aided drug design can be used to identify or design candidate compounds, such as inhibitors, agonists and antagonists, that bind to and/or modulate the activity of a protein serine/threonine phosphatase. Once identified and screened for binding capability and/or biological activity, these inhibitors/agonists/antagonists may be used therapeutically or prophylactically to block the receptor and therefore should be useful in preventing transplant rejection.

Candidate compounds that are identified or designed by computer methods to interact with one or more of the binding motifs identified on the catalytic subunit of a protein serine/threonine phosphates are potential modulators of the enzyme's activity and are therefore potential drug candidates. It is therefore possible to identify potential candidate compounds by consideration of the X-Ray crystallography data. Such data can be used in computational methods well known in the art to determine which residues are on the surface of the catalytic subunit representing the one or more binding motifs identified by the inventors and therefore potentially able to interact with other molecules in solution. In general, these techniques rely on graphic representations of the receptor and computer assisted manipulation of the graphic representation. The structural coordinate data stored in a machine-readable storage medium that is capable of displaying a graphical three-dimensional representation of the structure of the binding motif(s) or portion(s) thereof in a liganded and unliganded state can therefore be used in drug discovery. The structure coordinates of the candidate compound are used to generate a three-dimensional image that can be computationally fitted to the three-dimensional image of the enzyme, or a fragment thereof.

The process of rational drug described above is considered by the inventors of the present invention to be well known to skilled addressees in this area and it is felt that the description given above would be sufficient to explain the process to a skilled addressee. Nevertheless, in order to assist a lay reader we provide a brief description of computational design intended to detail how the data provided by the present invention can be utilised in this process. Various computational analyses are can be used to determine whether a molecule is sufficiently complementary to the target moiety or structure to be useful as a pharmaceutical agent. Such analyses may be carried out in current software applications, such as the Molecular Similarity application of QUANTA (Molecular Simulations Inc., Waltham, Mass.) version 3.3, and as described in the accompanying User's Guide, Volume 3 pages 134-135.

The Molecular Similarity application permits comparisons between different structures, different conformations of the same structure, and different parts of the same structure. The procedure used in Molecular Similarity to compare structures is divided into four steps:

1 ) load the structures to be compared;

2) define the atom equivalences in these structures; 3) perform a fitting operation; and

4) analyze the results.

Each structure is identified by a name. One structure is identified as the target (i.e., the fixed structure); all remaining structures are working structures (Ze., moving structures). When a rigid fitting method is used, the working structure is translated and rotated to obtain an optimum fit with the target structure. The fitting operation uses a least squares fitting algorithm which computes the optimum translation and rotation to be applied to the moving structure, such that the root mean square difference of the fit over the specified pairs of equivalent atom is an absolute minimum. This number, given in angstroms, is reported by QUANTA.

One skilled in the art may use one of several methods to screen chemical entities or fragments for their ability to associate with a target site. Again, these methods require no elucidation for the skilled person, but are described here for the benefit of the unskilled reader. The screening process begins by visual inspection of the target site on the computer screen (i.e., the binding motifs of the present invention or portions thereof), generated from a machine-readable storage medium. Selected fragments or chemical entitles may then be positioned in a variety of orientations, or docked, within that binding pocket as defined above. Docking may be accomplished using software such as Quanta and Sybyl, followed by energy minimization and molecular dynamics with standard molecular mechanics force fields, such as CHARMM and AMBER.

Specialized computer programs may also assist in the process of selected fragments or chemical entities. These include: 1. GRID (Goodford, 1985). GRID is available from Oxford University, Oxford, UK;

2. MCSS (Miranker et al., 1991 ). MCSS is available from Molecular Simulations, Burlington, Mass;

3. AUTODOCK (Goodsell, 1990). AUTODOCK is available from Scripps Research Institute, La JoIIa, Calif; and 4. DOCK (Kuntz, 1982). DOCK is available from University of California, San Francisco, Calif.

Once a suitable candidate compound or fragments thereof have been selected, they can be assembled into a single compound or complex. Assembly may be preceded by visual inspection of the relationship of the fragments to each other on the three- dimensional image displayed on a computer screen in relation to the structure coordinates of the target compound or site. This would be followed by manual model building using software such as Quanta or Sybyl.

Useful programs to aid one of skill in the art in connecting the individual chemical entities or fragments include:

1. CAVEAT (Bartlett, 1989). CAVEAT is available from the University of

California, Berkeley, Calif; 2. 3D Database systems such as MACCS-3D (MDL Information Systems, San

Leandro, Calif.); and 3. HOOK (available from Molecular Simulations Burlington, Mass.).

As the skilled reader will already know, instead of proceeding to build a ligand for the target in a step-wise fashion, one fragment or candidate compound at a time as described above, target-binding compounds may be designed as a whole or de novo. These methods include: 1. LUDI (Bohm, 1992). LUDI is available from the Biosym Technologies, San

Diego, Calif; 2. LEGEND (Nishibata, 1991 ). LEGEND is available from Molecular Simulations,

Burlington, Mass; and 3. LeapFrog (available from Tripos Associates, St. Louis. Mo.).

Other molecular modelling techniques may also be employed.

Once a compound has been designed or selected by such methods, the efficiency with which that compound can bind to a target site may be tested and optimized by computational evaluation. For example, an effective ligand will preferably demonstrate a relatively small difference in energy between its bound and free states; i.e. a small deformation energy of binding. Thus the most efficient ligand should preferably be designed with a deformation energy of binding of not greater than about 10kcal/mole, preferably, not greater than 7 kcal/mole. Ligands may interact with the target in more than one conformation that is similar in overall binding energy. In those cases, the deformation energy of binding is taken to be the difference between the energy of the free entity and the average energy of the conformations observed when the inhibitor binds to the protein.

A candidate compound designed or selected as binding to a target may be further computionally optimized so that in its bound states it would preferably lack repulsive electrostatic interaction with the target enzyme. Such non-complementary (e.g., electrostatic) interactions include repulsive charge-charge, dipole-dipole and charge- dipole interactions. Specifically, the sum of all electrostatic interactions between the ligand and the target, when the ligand is bound to the target, preferably makes a neutral or favourable contribution to the enthalpy of binding.

Once the candidate compound has been optimally selected or designed, as described above, substitutions may then be made in some of its atoms or side groups in order to improve or modify its binding properties. Generally initial substitutions are conservative, i.e., the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. It should, of course, be understood that components known in the art to alter conformation should be avoided. Such substituted chemical compounds may then be analyzed for efficiency of fit to the desired target site by the same computer methods described in detail, above. Again, all these facts are familiar to the skilled person. Another approach is the computational screening of small molecule data bases for chemical entities or compounds which can bind in whole, or in part, to a desired target. In this screening, the quality of fit of such entities to the binding site may be judged either by shape complementarity or by estimated interaction energy.

The computational analysis and design of molecules, as well as software and computer systems therefore, are described in U. S Patent No. 5,978,740 which is included herein by reference, including specifically, but not by way of limitation, the computer system diagram described with reference to and illustrated in Figure 3 thereof, as well as the data storage media diagram described with reference to and illustrated in Figure 4s and 5 thereof.

Pursuant to the present invention, such stereochemical complementarity is characteristic of a molecule which matches intra-site surface residues lining the binding regions identified herein. By "match" we mean that the identified portions interact with the surface residues, for example, via hydrogen bonding or by enthalpy/entropy-reducing Van der Waals interactions which promote desolvation of the biologically active compound within the site, in such a way that retention of the biologically active compound within the groove is energetically favoured.

Thus, there are two preferred approaches to designing a molecule according to the present invention, which complements the shape of the binding sites. In the first of these, the geometric approach, the number of internal degrees of freedom, and the corresponding local minima in the molecular conformation space, is reduced by considering only the geometric (hard-sphere) interactions of two rigid bodies, where one body (the active site) contains "pockets" or "grooves" which form binding sites for the second body (the complementing molecule, as ligand). The second approach entails an assessment of the interaction of different chemical groups ("probes") with the binding motif(s) of the present invention at sample positions within and around the site, resulting in an array of energy values from which three-dimensional contour surfaces at selected energy levels can be generated.

The geometric approach is illustrated by Kuntz et al. (Kuntz, I. D. et al. J. MoI. Biol. 161 :269-288, 1982; the contents of which are hereby incorporated by reference), whose algorithm for the design of candidate compounds {e.g., ligands) is implemented in a commercial software package distributed by the Regents of the University of California and further described in a document, provided by the distributor, entitled Overview of the DOCK Package, Version 1.0,", the contents of which are hereby incorporated by reference. Pursuant to the Kuntz algorithm, the shape of the cavity represented by the substrate/ligand binding site is defined as a series of overlapping spheres of different radii. One or more extant databases of crystallographic data, such as the Cambridge Structural Database System maintained by Cambridge University (University Chemical Laboratory, Lensfield Road, Cambridge CB2 1 EW, U.K.) and the Protein Data Bank maintained by the Research Collaboratory for Structural Bioinformatics (RCSB; http//www.rcsb.org/index.html) is then searched for molecules which approximate the shape thus defined.

Programs suitable for pharmacophore selection and design include: DISCO (Abbott Laboratories, Abbott Park, IL), Catalyst (Bio-CAD Corp., Mountain View, CA), and ChemDBS-3D (Chemical Design Ltd., Oxford, U.K.).

Databases of chemical structures are available from a number of sources including Cambridge Crystallographic Data Centre (Cambridge, U.K.) and Chemical Abstracts Service (Columbus, OH).

Those skilled in the art will recognize that the design of a mimetic compound may require slight structural alteration or adjustment of a chemical structure designed or identified using the methods of the invention. This aspect of the invention may be implemented in hardware or software, or a combination of both. However, in one embodiment, the invention is implemented in computer programs executing on programmable computers each comprising a processor, a data storage system (including volatile and non-volatile memory and/or storage elements), at least one input device, and at least one output device. Program code is applied to input data to perform the functions described above and generate output information. The output information is applied to one or more output devices, in known fashion. The computer may be, for example, a personal computer, microcomputer, or work station of conventional design.

Each program is preferably implemented in a high level procedural or object-oriented programming language to communicate with a computer system. However, the programs can be implemented in assembly or machine language, if desired. In any case, the language may be compiled or interpreted language.

Each such computer program is preferably stored on a storage medium or device

(e.g., ROM or magnetic diskette) readable by a general or special purpose programmable computer, for configuring and operating the computer when the storage media or device is read by the computer to perform the procedures described herein. The inventive system may also be considered to be implemented as a computer-readable storage medium, configured with a computer program, where the storage medium so configured causes a computer to operate in a specific and predefined manner to perform the functions described herein.

In one embodiment of the present invention, the in silico screening methods are implemented in hardware or software, or a combination of both. The methods may be implemented in computer programs executing on programmable computers each comprising a processor, a data storage system including volatile and non-volatile memory and/or storage elements, at least one input device, and at least one output device. The computer program may also be implemented in a high level procedural or object-oriented programming language to communicate with a computer system. The computer program may also be stored on a storage medium or device readable by a general or special purpose programmable computer, for configuring and operating the computer when the storage media or device is read by the computer to perform a method according to the invention.

In another embodiment of the present invention, the candidate compound(s) identified or designed by the methods of the present invention as herein described have a high affinity for the selected target site (i.e., binding motif). For in silico screening using computer modelling systems, the affinity constant is preferably <1 μM, more preferably < 1 nM.

In one embodiment, the three dimensional structure of the catalytic subunit of the protein serine/threonine phosphatase is provided by using the atomic coordinates of amino acids of protein serine/threonine phosphatase according to those publicly disclosed in the RCSB Protein Databank under accession 2iae

(http://www.rcsb.org/pdb/files/2iae.pdb); as accessed 7 December 2007, and the step of identifying a candidate compound for binding to one or more binding motifs on the catalytic subunit is performed using a fitting operation between the three-dimensional structure of the one or more binding motifs and the candidate compound. In a certain embodiment, the candidate compound demonstrates a docking score of at least about

-2.5, and preferably less than about -7.5. In yet another embodiment of the present invention, the one or more binding motifs are represented by the amino acid residues selected from the group consisting of: (a) D57, H59, D85, R89, N117, H118, H167, R214, H241;

(b) D57, H59, D85, N117, H167, H241;

(C) 162, 163,235,236,250,254,278,282,284;

(d) N117,H118,S120,R121,Q122,l123,T124,E188,V189,P190,H191,G192, C196,W200,A216,G217;

(e) I14,N18,M66,F69,R7O,G73,K74,S75,Y8O,L99,L1O3;

(f) F164,L166,G169,L17O,S171,I174,I18O,L198,W2O9,F228;

C) L31,A35,L39,L100,V101,K104,V105,l113,L149,L153;

(k) D57,V58,Q61 ,D64,L65,L68,S261 ,A262,P263,F289;

(I) C55,G56,D57,V58,H59,L81,F82,M83,G84,D85,Y86,L100,L114,S261;

(m) D57,H59,D85,R89,N117,H118,l123,Y127,W200,R214,H241,F260,Y265; (n) V28,K29,C32,E33,K36,K144,Y145,D148,L149;

(o) I14,S75,P76,L1O3,R1O6,Y1O7,I111;

(p) L39,T40,D151,Y152,L153,R185,L186,Q187;

(q) H191,E192,G193,C196,D197,A216,G217,Y218;

(r) Y107,R108,E109,R110; (s) P51,V52,T53,D77,T78,N79,Y80,M276,E277,L278,D279 ;

(t) D131,L134,R135,Y307,F308;

(u) V244,M245,E246,G247,Y248,N249,W250;

(v) W200,S201,D202,S212,P213,R214,A216,G217,Y218,T219,F220;

(w) R89,R214,H241,Q242,L243,F260,Y265; (x) F6,W13,S30,L31,E33,K34,E37;

(y) L10,D11,l14,E15,P76,R106,Y107;

(z) G90,Y91,Y92,S93,G128,F129,D131,E132,R135,K136;

(aa) L17O,S171,I174,D175,W2O9,I224,T227,F228;

(dd) P203,D204,D205,R206,G207,G221,Q222,D223,Q242,N249,C251,H252,D253;

(ee) L183,D184,R185,Q187,P190,P194,H195;

(ff) Y267,P291,A292,R294;

(gg) P203, R239,Q242, L243,V244, N249J258; (hh) E67,l71,F289,D290,P291,A292,P293;

(ii) E33,K36,E37; Cj) V5,F6,T7,K8,E9;

(kk) I21 1 ,S212,G215,A216,G217,Y218;

(II) E226,N229,H230,L234,N255;

(mm) T176,L177,D178,R181 ,N232; (nn) H63,H66,E67,R70,A292,P293,R294;

(oo) H63,Y92,Y267,R294,R295;

(PP) D202, P203, D204, P213.T219, F220,Q242;

(qq) V300,R303,T304,P305;

(rr) H63,P293,R294,R295; (SS) T40,N44,L183,D184,R185,L186;

(tt) E42,Q46,V48,K104,R108,E109,l1 11 ,T112;

(UU) D204,R206,G210,l21 1 ,S212,P213,T219;

(W) D204,R206,l211 ,P213,T219; and

Y265;

(yy) N1 17, S120, R121 , Q122, 1123, Y127, E188, V189, P190, H191 , W200;

(ZZ) D204, D205, C221 , Q222, G251 , H252, D253;

(aaa) R89, R214; (bbb) H191. Q122;

(ccc) E188, R121 ;

(ddd) H252, D253; and

(eee) D290.

In one embodiment, the binding motif is a component of a binding complex, the binding complex comprising an accessory atom, ion or molecule. Preferably, the accessory ion is Mn2+.

The methods of identifying candidate compounds in accordance with the present invention may also involve contacting a candidate compound with a binding motif on the catalytic subunit of a protein serine/threonine phosphatase for a sufficient time to allow for binding to, activation or inhibition of the enzyme by the candidate compound.

The period of contact with the candidate compound being tested can be varied depending on the result being measured, and can be determined by one of skill in the art. For example, for binding assays, a shorter time of contact with the candidate compound being tested is typically suitable, than when activation is assessed. As used herein, the term " contact period' generally refers to the time period during which the catalytic subunit of the protein serine/threonine phosphatase is in contact with the compound being tested. The term "incubation period", as used herein, generally refers to the entire time during which cells expressing the catalytic subunit of the protein serine/threonine phosphatase are allowed to grow prior to evaluation, and can be inclusive of the contact period. Thus, the incubation period includes all of the contact period and may include a further time period during which the compound being tested is not present but during which growth is continuing (in the case of a cell based assay) prior to scoring. The incubation time for growth of cells can vary but is sufficient to allow for the binding of candidate compound, activation of the enzyme activity or signal transduction pathways associated with the enzyme activity, and/or inhibition of the enzyme activity. It will be recognized that shorter incubation times are preferable because compounds can be more rapidly screened. In one embodiment, the incubation time is between about 1 minute to about 48 hours.

In one embodiment, a cell-based assay is conducted under conditions which are effective to screen for candidate compounds useful in the method of the present invention. Effective conditions include, but are not limited to, appropriate media, temperature, pH and oxygen conditions that permit the growth of the cell that expresses the receptor. An appropriate, or effective, medium refers to any medium in which a cell that naturally or recombinantly expresses a catalytic subunit of a protein serine/threonine phosphatase, when cultured, is capable of cell growth and expression of the catalytic subunit. Such a medium is typically a solid or liquid medium comprising growth factors and assimilable carbon, nitrogen and phosphate sources, as well as appropriate salts, minerals, metals and other nutrients, such as vitamins. Culturing is carried out at a temperature, pH and oxygen content appropriate for the cell. Such culturing conditions are within the expertise of one of ordinary skill in the art.

In one embodiment, a BIAcore machine can be used to determine the binding constant of a complex between the catalytic subunit and a ligand (e.g., sodium selenate) in the presence and absence of the candidate compound. The dissociation constant for the complex can be determined by monitoring changes in the refractive index with respect to time as buffer is passed over the chip (see O'Shannessy et al. Anal. Biochem. 212:457-468, 1993; and Schuster et al., Nature 365:343-347, 1993).

Other suitable assays for measuring the binding of a candidate compound to a catalytic subunit of a protein serine/threonine phosphatase, and or for measuring the ability of a candidate compound to affect the binding of a catalytic subunit of a protein serine/threonine phosphatase to a ligand such as sodium selenate include, for example, immunoassays such as enzyme linked immunoabsorbent assays (ELISA) and radioimmunoassays (RIA), as well as cell-based assays including, cytokine secretion assays, or intracellular signal transduction assays that determine, for example, protein or lipid phosphorylation.

Modulation of Protein Serine/Threonine Phosphatase Activity

In another embodiment of the present invention, the method includes obtaining the identified candidate compound, contacting the compound with a protein serine/threonine phosphatase, or with an analogue, a homolog or a fragment thereof, and determining the ability of the compound to modulate protein serine/threonine phosphatase activity.

In one embodiment, protein serine/threonine phosphatase activity is determined by measuring the level of enzyme present in cells by Western blotting. For example, protein levels of the enzyme can be measured in fibroblasts using an antibody (e.g., an anti-PP2A antibody; Biosource). Levels of a different protein can also be measured in the same sample as a reference protein for normalization. Examples of possible reference proteins include, but are not limited to, annexin-ll or actin. In another embodiment, the level of protein serine/threonine phosphatase activity in AD and AC cells is assayed according to a procedure (Pierce Biotechnology) using p-nitrophenyl phosphate (PNPP) as the substrate. For instance, the enzyme activity assays are carried out in a 96-well microplate. The reaction is initiated by adding about 10 μl of each AC or AD cell lysate into about 90 μl of reaction mixture, incubated at about 30⁰C for about 15 minutes, and measured in a BioRad microplate reader at a wavelength of 420 nM. After subtraction of values from reactions in which about 10 nM of an enzyme inhibitor such as okadiac acid is present, the activity of the enzyme is calculated according to a standard curve produced by a series of known concentrations of purified enzyme. In one embodiment, ELISA is performed according to the following procedures: 1 ) Add fibroblast cell lysates after treatment in duplicates or triplicates to a 96-well microplate that is previously coated with an anti-Erk antibody. 2) Incubate samples in microplate wells at room temperature for about 2 hours. 3) Aspirate samples and wash wells with a phosphate buffered saline (PBS)-based washing buffer. 4) Add working dilution of an anti phospho-Erkl/2, or an anti-regular Erkl/2 antibody to each well, and incubate at room temperature for about 1 hour. 5) Aspirate and wash well with washing buffer. 6) Add a working dilution of a secondary antibody conjugated with horseradish peroxidase (HRP) to each well and incubate well at room temperature for about 30 min. 7) Aspirate and wash well with washing buffer. 8) Add stabilized Chromogen such as diaminobenzidine (DAB) and incubate at room temperature for about 30 min. 9) Add stop solution and measure the absorbance at 450 nm. Phosphorylation of Erkl/2 is assessed after normalization: NR = AP/AR. Where NR = the normalized ratio; Ap is absorbance values for phospho-Erkl/2; and A_R is absorbance for the total .. (regular) Erkl/2.

Candidate Compounds

In another aspect of the present invention, there is provided a candidate compound isolated or obtained by the methods of the present invention as herein described.

Suitable candidate compounds to test using the method of the present invention include, but are not limited to, proteins, peptides or other organic molecules, and inorganic molecules. Suitable organic molecules include small organic molecules. Peptides refer to small molecular weight compounds yielding two or more amino acids upon hydrolysis. A polypeptide is comprised of two or more peptides. As used herein, a protein is comprised of one or more polypeptides. Preferred therapeutic compounds to design include peptides composed of "L" and/or "D" amino acids that are configured as normal or retroinverso peptides, peptidomimetic compounds, small organic molecules, or homo- or hetero-polymers thereof, in linear or branched configurations.

In one embodiment of the present invention, the compound isolated or obtained by the methods of the present invention includes a tetrahedral structure that has a net negative charge at physiological pH. Compounds may include sulphate or selenium. In a certain embodiment, the compound includes a sulphate (SO₄ ^2"), a selenate (SeO₄ ^2") or a pharmaceutically acceptable salt thereof. In a certain embodiment, a compound that is identified by the methods of the present invention originates from a compound having chemical and/or stereochemical complementarity with selenate which binds to the binding motifs of the catalytic subunit of a protein serine/threonine phosphatase such as PP1 , PP2A and PP5 and acts as an agonist of protein serine/threonine phosphatase activity. Such compounds having chemical and/or stereochemical complementarity with selenate include a selenone including, but not limited to SeO₂(CH₃)₂, SeO₃CH₃ ^", SeO₂CH₃ ^" and pharmaceutically acceptable salts thereof. Other examples of candidate compounds are given in Figure 3. With reference to Figure 3, substituent R is typically any moiety that will not adversely affect the ability of the compound to bind to one or more binding motifs of the catalytic subunit of a protein serine/threonine phosphatase and/or adversely affect the ability of that compound to modulate protein serine/threonine phosphatase activity.

Candidate compounds are typically capable of modulating the activity of a protein serine/threonine phosphatase; that is, enhancing (agonists) or inhibiting (antagonists) enzyme activity.

As used herein, the term "agonist' generally refers to any compound that interacts with a protein serine/threonine phosphatase and elicits an observable response. More particularly, an agonist can include, but is not limited to, a protein (including an antibody), a peptide, a nucleic acid or any suitable product of drug design {e.g., a mimetic) which is characterized by its ability to agonize (e.g., stimulate, induce, increase, enhance) the biological activity of a naturally occurring protein serine/threonine phosphatase in a manner similar to a natural agonist (e.g., by interaction/binding with and/or direct or indirect activation of the protein serine/threonine phosphatase, including by stabilizing the interaction of the protein serine/threonine phosphatase with a natural ligand). An "antagonist' refers to any compound which inhibits the effect of protein serine/threonine phosphatase agonist, as described above. More particularly, a protein serine/threonine phosphatase antagonist is capable of associating with the enzyme such that the biological activity of the receptor is decreased (e.g., reduced, inhibited, blocked, reversed, altered) in a manner that is antagonistic (e.g., against, a reversal of, contrary to) to the action of a natural agonist on the receptor. Agonists (i.e., stimulatory compounds) identified using the present methods include compounds that exhibit improved binding to a catalytic subunit of a protein serine/threonine phosphatase when compared with the ability of a natural ligand of the enzyme, and also include compounds that enhance the binding of a natural ligand to the catalytic subunit of the enzyme. Agonists may also be identified by their ability to: (1 ) bind to, or otherwise interact with, a catalytic subunit of a protein serine/threonine phosphatase at a higher level than, for example, a natural ligand of the enzyme and/or; (2) enhance binding of the catalytic subunit of the enzyme to its natural ligand. Another suitable agonist compound of the present invention can include a compound that binds to a catalytic subunit of a protein serine/threonine phosphatase in the absence of a natural ligand of the enzyme in such a manner that enzyme-mediated cellular signal transduction is stimulated.

Suitable antagonist (i.e., inhibitory) compounds that may be identified using the methods of the present invention include compounds that interact directly with the catalytic subunit of the protein serine/threonine phosphatase, thereby inhibiting the binding of a natural ligand to the enzyme, by either blocking the binding motifs on the catalytic subunit of the enzyme (referred to herein as substrate analogs) or by modifying other regions of the protein serine/threonine phosphatase such that the natural ligand cannot bind to the enzyme (e.g., by allosteric interaction). As used herein, the term "substrate analogs" refers to compounds that interacts with (e.g., binds to, associates with, modifies) the ligand binding motif of a catalytic subunit of a protein serine/threonine phosphatase. A substrate analog can, for example, comprise a chemical compound that mimics the binding motif of the natural catalytic subunit of a protein serine/threonine phosphatase, or that binds specifically to the ligand binding motif of the catalytic subunit of the enzyme but does not mimic the complementary binding portion of the natural ligand. An inhibitory compound of the present invention can also include a compound that essentially mimics at least a portion of a catalytic subunit of a protein serine/threonine phosphatase that binds to a natural ligand (referred to herein as a peptidomimetic compound).

Methods of identifying candidate compounds and selecting compounds that bind to, activate or inhibit protein serine/threonine phosphatase activity have been previously described herein. Candidate compounds can be synthesized using techniques known in the art, and depending on the type of compound. Synthesis techniques for the production of non-protein compounds, including organic and inorganic compounds are well known in the art.

For smaller peptides, chemical synthesis methods are preferred. For example, such methods include 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. Such methods are well known in the art and may be found in general texts and articles in the area such as: Merrifield, 1997, Methods Enzymol. 289:3-13; Wade et al., 1993, Australas Biotechnol. 3(6):332- 336; Wong et al, 1991 , Expehentia 47(11-12):1123-1129; Carey et ai, 1991 , Ciba Found Symp. 158:187-203; Plaue et al., 1990, Biologicals 18(3): 147-157; Bodanszky, 1985, Int. J. Pept. Protein Res. 25(5):449-474; or H. Dugas and C. Penney, Bioorganic Chemistry (1981 ) at pages 54-92, all of which are incorporated herein by reference in their entirety. For example, peptides may be synthesized by solid-phase methodology utilizing a commercially available peptide synthesizer and synthesis cycles supplied by the manufacturer. One skilled in the art recognizes that the solid phase synthesis could also be accomplished using the FMOC strategy and a TFA/scavenger cleavage mixture.

If larger quantities of a protein are desired, or if the protein is a larger polypeptide, the protein can be produced using recombinant DNA technology. A protein can be produced recombinantly by culturing a cell capable of expressing the protein {i.e., by expressing a recombinant nucleic acid molecule encoding the protein) under conditions effective to produce the protein, and recovering the protein. Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production. An effective medium refers to any medium in which a cell is cultured to produce the protein. Such medium typically comprises an aqueous medium having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. Recombinant cells (i.e., cells expressing a nucleic acid molecule encoding the desired protein) can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art. Such techniques are well known in the art and are described, for example, in Sambrook et al., 1988, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. or Current Protocols in Molecular Biology (1989) and supplements.

Pharmaceutical Compositions and Methods of Treatment Once a compound has been identified that is capable of modulating the activity of protein serine/threonine phosphatase, as herein described, the compound can administered to a patient to treat a condition associated with the activity of a protein serine/threonine phosphatase, including, but not limited to a hyperproliferative disorder (e.g. cancer), a neurodegenerative disease and a disorder associated with aberrant angiogenesis. As discussed supra, it is not necessary for the condition to be caused by an aberrant or lower than normal level of phosphatase activity.

The term "neurodegenerative disease" as used herein, refers to a neurological disease characterised by loss or degeneration of neurons. Neurodegenerative diseases include neurodegenerative movement disorders and neurodegenerative conditions relating to memory loss and/or dementia. Neurodegenerative diseases include tauopathies and α-synucleopathies. Examples of neurodegenerative diseases include, but are not limited to, presenile dementia, senile dementia, Alzheimer's disease, Parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy (PSP), Pick's disease, primary progressive aphasia, frontotemporal dementia, corticobasal dementia, Parkinson's disease, dementia with Lewy bodies, Down's syndrome, multiple system atrophy, amyotrophic lateral sclerosis (ALS) and Hallervorden-Spatz syndrome.

In some embodiments, the neurological disorder is selected from the group consisting of Creutzfeldt-Jakob disease, Huntington's disease, stroke, cerebral ischaemia, dementia associated with stroke or cerebral ischaemia, dementia associated with HIV, disorders associated with excitotoxicity, epilepsy, seizures, schizophrenia, multiple sclerosis, acute brain trauma (severe traumatic brain injury) and motor neurone disease.

In specific embodiments, the cancer may be selected from the group consisting of brain, lung, liver, spleen, kidney, lymph node, small intestine, pancreas, blood cells, colon, stomach, breast, endometrium, prostate, testicle, ovary, skin, head and neck, esophagus, bone marrow and blood cancer. In more specific embodiments, the cancer is a lung cancer, colon cancer, breast cancer or cervical cancer. Hyperproliferative disorders refers to excess cell proliferation, relative to that occurring with the same type of cell in the general population and/or the same type of cell obtained from a patient at an earlier time. The term denotes malignant as well as non- malignant cell populations. Such disorders have an excess cell proliferation of one or more subsets of cells, which often appear to differ from the surrounding tissue both morphologically and genotypically. The excess cell proliferation can be determined by reference to the general population and/or by reference to a particular patient, e.g. at an earlier point in the patient's life. Hyperproliferative cell disorders can occur in different types of animals and in humans, and produce different physical manifestations depending upon the affected cells.

Other hyperproliferative disorders that may be associated with altered activity of phosphorylation modifying enzyme(s) include a variety of conditions where there is proliferation and/or migration of smooth muscle cells, and/or inflammatory cells into the intimal layer of a vessel, resulting in restricted blood flow through that vessel, i.e. neointimal occlusive lesions. Occlusive vascular conditions of interest include atherosclerosis, graft coronary vascular disease after transplantation, vein graft stenosis, peri-anastomatic prosthetic graft stenosis, restenosis after angioplasty or stent placement, and the like. Other disorders and conditions of interest relate to epidermal hyperproliferation, tissue remodelling and repair. For example, the chronic skin inflammation of psoriasis is associated with hyperplastic epidermal keratin ocytes.

As discussed supra, non-tumor diseases or disorders associated with aberrant angiogenesis include conditions improved by removal, inhibition or reduction of angiogenic growth factors such as clinical conditions characterised by excessive vascular endothelial cell proliferation, vascular permeability, edema or inflammation comprising macular degeneration, especially age-related macular degeneration comprising wet macular degeneration and dry macular degeneration; retinopathies such as diabetic retinopathy, ischaemic retinal vein occlusion and retinopathy of prematurity; endometriosis, restenosis, psoriasis and rheumatoid arthritis, brain edema associated with injury, stroke or tumor, edema associated with inflammatory conditions such as psoriasis, and arthritis comprising rheumatoid arthritis, asthma, generalised edema associated with burns, ascites and pleural effusion, especially macular degeneration, diabetic retinopathy, endometriosis and restenosis.

A composition to be administered to a patient, such as in this embodiment, generally includes the compound identified by the methods of the present invention and a carrier, such as a pharmaceutically acceptable carrier. Exemplary compositions are described herein supra.

A composition which includes a compound identified according to the present methods can be delivered to a cell culture or patient by any suitable method. Selection of such a method will vary with the type of compound being administered or delivered {i.e., protein, peptide, nucleic acid molecule, mimetic, or other type of compound), the mode of delivery {i.e., in vitro, in vivo, ex vivo) and the goal to be achieved by administration/delivery of the compound or composition. According to the present invention, an effective administration protocol (i.e., administering a composition in an effective manner) comprises suitable dose parameters and modes of administration that result in delivery of a composition to a desired site (i.e., to a desired cell) and/or in the desired regulatory event (e.g., to potentiate protein serine/threonine phosphatase activity).

It will be apparent to one of skill in the art that the number of doses of a compound identified by the methods of the present invention (as herein described) as capable of modulating protein serine/threonine phosphatase activity administered to a patient in need thereof will depend on the severity of the condition and the response of an individual to the treatment. It is within the scope of the present invention that a suitable number of doses, as well as the time periods between administrations, includes any number required to cause regression of the condition.

In another embodiment of the present invention, a compound identified by the methods of the present invention (as herein described) may also be employed in conjunction with administration to the patient of one or more therapeutic compounds specific for their particular condition. Another embodiment of the present invention relates to an antibody that selectively binds to one or more of the binding motifs on the catalytic subunit of a protein serine/threonine phosphatase. The present inventors have provided suitable target sites (binding motifs) for the design and identification of antibodies.

According to the present invention, the phrase "selectively binds to" refers to the ability of an antibody, antigen binding fragment or binding partner of the present invention to preferentially bind to specified proteins {e.g., the recited portions of the protein serine/threonine phosphatase). More specifically, the phrase "selectively binds" refers to the specific binding of one protein to another {e.g., an antibody, fragment thereof, or binding partner to an antigen), wherein the level of binding, as measured by any standard assay {e.g., an immunoassay), is statistically significantly higher than the background control for the assay. For example, when performing an immunoassay, controls typically include a reaction well/tube that contain antibody or antigen binding fragment alone (i.e., in the absence of antigen), wherein an amount of reactivity (e.g., non-specific binding to the well) by the antibody or antigen binding fragment thereof in the absence of the antigen is considered to be background. Binding can be measured using a variety of methods standard in the art including enzyme immunoassays (e.g., ELISA), immunoblot assays, etc.

Limited digestion of an immunoglobulin with a protease may produce two fragments. An antigen binding fragment is referred to as an Fab, an Fab', or an F(ab')₂ fragment. A fragment lacking the ability to bind to antigen is referred to as an Fc fragment. An Fab fragment comprises one arm of an immunoglobulin molecule containing a L chain (V_L + C_L domains) paired with the V_H region and a portion of the C_H region (CH 1 domain). An Fab' fragment corresponds to an Fab fragment with part of the hinge region attached to the CH 1 domain. An F(ab')₂ fragment corresponds to two Fab' fragments that are normally covalently linked to each other through a di-sulfide bond, typically in the hinge regions.

As discussed above, a composition, particularly a pharmaceutical composition, of the present invention generally includes the therapeutic compound (e.g., the compound identified by the structure based identification method) and a carrier, and perhaps also a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers and exemplary methods of administration of therapeutic compositions of the present invention have been described in detail above with regard to the administration of an candidate compound to treat a condition associated with aberrant protein serine/threonine phosphatase activity. Such carriers and administration protocols are applicable to this embodiment.

Optimization of Candidate Compounds

In yet another embodiment of the present invention, chemical analogs or derivatives of the candidate compounds is also provided, as well as testing of these analogs or derivatives for binding to or modulating the activity of a protein serine/threonine phosphatases. The structure activity relationship (SAR) can provide a great deal of information regarding the nature of ligand-receptor interactions, but no detailed information about the location and actual chemical nature of the binding site in the target protein is provided. A number of closely related chemical structures may be used to direct the orientation of the ligand within the putative binding cavity and to determine what part of the ligand is involved in binding to the receptor (see, e.g., Mattos et al. Struct. Biol., 1995 1 :55-58).

Once a potential modulating compound has been identified in accordance with the methods of the present invention {e.g., by comparing the activity of the compound in an enzyme assay to the activity of an appropriate standard), structure-based design methods can be used to optimize the inhibitor. Using drug-like molecules pre- screened in silico with computer models of the active site can enhance the high- throughput screen for lead compounds.

For example, the candidate compound and the protein serine/threonine phosphatase can be crystallized as a complex and the crystal structure of the complex can be determined. The structural information obtained from the crystal structure can then be used to formulate pharmacophore hypotheses. This process may be repeated to provide increasingly potent and specific enzyme modulators.

A computational pharmacophore search can be carried out using X-ray crystallographic structural information to generate a computational model. Commercially available compounds can be docked and selected for screening using the docking score as one, but not necessarily the only, element for consideration. Additional analogs can be bought or synthesized, and then screened. Experiments with these analogs can be used to confirm the hypothesis from the previous screening experiments or to suggest new hypotheses that can similarly be tested by repeating the process. In some cases, alternative templates can be identified and compounds based on these templates can be bought or synthesized to test the new hypotheses. It can be desirable to identify pharmaceutically relevant templates, and/or templates that best test complementary binding hypotheses. In each case, the compounds are typically screened against the enzyme target and also tested for in vitro antibacterial activity.

Moreover, molecular modelling techniques are known in the art, including both hardware and software appropriate for creating and utilizing models of receptors and enzyme conformations.

Numerous computer programs are available and suitable for rational drug design and the processes of computer modeling, model building, and computationally identifying, selecting and evaluating potentially useful compounds in the methods described herein. These include, for example, GRID (available form Oxford University, UK), MCSS (available from Accelrys, Inc., San Diego, Calif.), AUTODOCK (available from Oxford Molecular Group), FLEX X (available from Tripos, St. Louis. MO), DOCK (available from University of California, San Francisco), CAVEAT (available from University of California, Berkeley), HOOK (available from Accelrys, Inc., San Diego, Calif.), and 3D database systems such as MACCS-3D (available from MDL Information Systems, San Leandro, Calif.), UNITY (available from Tripos, St. Louis. MO), and CATALYST (available from Accelrys, Inc., San Diego, Calif.). Potential antimicrobial compounds may also be computationally designed "de novo" using such software packages as LUDI (available from Biosym Technologies, San Diego, Calif.), LEGEND (available from Accelrys, Inc., San Diego, Calif.), and LEAPFROG (Tripos Associates, St. Louis, Mo.). Compound deformation energy and electrostatic repulsion, may be evaluated using programs such as GAUSSIAN 92, AMBER, QUANTA/CHARMM, AND INSIGHT II/DISCOVER. These computer evaluation and modeling techniques may be performed on any suitable hardware including for example, workstations available from Silicon Graphics, Sun Microsystems, and others. These techniques, methods, hardware and software packages are representative and are not intended to be comprehensive listing. Other modeling techniques known in the art may also be employed in accordance with this invention. See for example, N. C. Cohen, Molecular Modeling in Drug Design, Academic Press (1996) (and references therein), and software identified at various internet sites.

Crystal Structures In another aspect of the present invention, there is provided a protein serine/threonine phosphatase crystal comprising a ligand including (but not limited to) sulphate or selenate in complex with the catalytic subunit of the phosphatase as disclosed, for example, in Cho and Xu, "Crystal structure of a protein phosphatase 2A heterotrimeric holoenzyme" Nature 445:53-57, 2007 and as publicly disclosed in the RCSB Protein Databank under accession 2iae (http://www.rcsb.org/pdb/files/2iae.pdb); as accessed

7 December 2007. Such crystals may be formed according to the methodology described in Cho and Xu (ibid).

One embodiment of the present invention includes a representation, or model, of the three dimensional structure of the catalytic subunit of a protein serine/threonine phosphatase, such as a computer model. A computer model of the present invention can be produced using any suitable software program, comprising MOLSCRIPT 2.0

(Avatar Software AB, Heleneborgsgatan 21 C, SE-1 1731 Stockholm, Sweden), the graphical display program O (Jones et al., Acta Crystallography, vol. A47:110, 1991 ), the graphical display program GRASP or the graphical display program INSIGHT.

Suitable computer hardware useful for producing an image of the present invention is known to those of skill in the art (e.g., a Silicon Graphics Workstation).

A representation, or model, of the three dimensional structure of the complex structure for which a crystal has been produced can also be determined using techniques which include molecular replacement or SIR/MIR (single/multiple isomorphous replacement).

Methods of molecular replacement are generally known by those of skill in the art

(see, for example, Brunger, Meth. Enzym., 276: 558-580, 1997; Navaza and

Saludjian, Meth. Enzym., 276:581-594, 1997; Tong and Rossmann, Meth. Enzym., 276:594-61 1 , 1997; and Bentley, Meth. Enzym., 276:611-619, 1997, each of which are incorporated by reference herein in their entirety) and are performed in a software program including, for example, AmoRe (CCP4, Acta Cryst. D50, 760-763 (1994) or

XPLOR. Briefly, X-ray diffraction data is collected from the crystal of a crystallized target structure. The X-ray diffraction data is transformed to calculate a Patterson function. The Patterson function of the crystallized target structure is compared with a

Patterson function calculated from a known structure (referred to herein as a search structure). The Patterson function of the crystallized target structure is rotated on the search structure Patterson function to determine the correct orientation of the crystallized target structure in the crystal. The translation function is then calculated to determine the location of the target structure with respect to the crystal axes. Once the crystallized target structure has been correctly positioned in the unit cell, initial phases for the experimental data can be calculated. These phases are necessary for calculation of an electron density map from which structural differences can be observed and for refinement of the structure. Preferably, the structural features of the search molecule are related to the crystallized target structure.

As used herein, the term "modef generally refers to a representation in a tangible medium of the three dimensional structure of a protein, polypeptide or peptide. For example, a model can be a representation of the three dimensional structure in an electronic file, on a computer screen, on a piece of paper (i.e., on a two dimensional medium), and/or as a ball-and-stick figure. Physical three-dimensional models are tangible and include, but are not limited to, stick models and space-filling models. The phrase "imaging the model on a computer screen" refers to the ability to express (or represent) and manipulate the model on a computer screen using appropriate computer hardware and software technology known to those skilled in the art. Such technology is available from a variety of sources including, for example, Evans and Sutherland, Salt Lake City, Utah, and Biosym Technologies, San Diego, Calif. The phrase "providing a picture of the mode? refers to the ability to generate a "hard copy" of the model. Hard copies include both motion and still pictures. Computer screen images and pictures of the model can be visualized in a number of formats including space-filling representations, .alpha, carbon traces, ribbon diagrams and electron density maps.

As used in this specification the term "structure coordinates" refers to Cartesian coordinates derived from mathematical equations related to the patterns obtained on diffraction of a monochromatic beam of x-rays by the atoms (scattering centres) of the crystal analysed. The diffraction data are used to calculate an electron density map of the repeating unit of the crystal. The electron density maps are then used to establish the positions of the individual atoms of the molecule making up the crystal.

As used herein the term "structural similarity means having a similar conformational structure. As used herein the term "homologue" means a protein having the same gross structural features as the parent protein but differing in function.

The present invention is further predicated at least in part on the surprising finding that some compounds can selectively activate or enhance the activity of some forms of PP2A but not others. This may account therefore for the diversity of the phosphatases and their ability to generate their specificity for different target proteins despite there being about 50 different phosphatase genes in the genome. It is also surprising that specific forms of the PP2A can be activated since all forms of the PP2A possess the same conserved catalytic domain.

The specific form of PP2A that now shows that it can be activated apart from others is the PP2AB form.

Accordingly, in a first aspect of the present invention there is provided a method of screening to identify a compound that selectively regulates phosphatase activity of a form of PP2A, said method comprising: contacting a sample containing PP2AB with a compound to be screened; and determining a differential effect of the compound on a PP2AB form of PP2A in the presence and absence of the compound.

As discussed supra, the phosphorylation state of key proteins is crucial in many cellular processes and depends on the precisely orchestrated balance of protein kinases and phosphatase activities.

The predominant form of PP2A in cells has a heterotrimeric subunit structure, consisting of a core dimer of approx 36 kDa conserved catalytic and approx 65 kDa conserved scaffold subunits (subunits C and A, respectively) complexed to a third variable subunit. Variable subunits are encoded by three multigene families (B, B', B").

A significant portion of the ABC isoform of PP2A is associated with neuronal microtubules, implicating PP2A in the regulation of the phosphorylation state of microtubule-associated proteins (MAPS), such as tau. PP2A containing the B regulatory subunits, but not other forms of PP2A (i.e. B' and B") has been shown to bind and potently de-phosphorylate tau in vitro. Furthermore, inhibiting the ABC isoform of PP2A induces hyperphosphorylation of tau, dissociation of tau from microtubules and loss of tau-induced microtubule stabilization. It has recently been shown that PP2A accounts for approximately 71 % of the total tau phosphatase activity of human brain. The total phosphatase activity and the activities of PP2A toward tau are significantly decreased in brains of Alzheimer's disease (AD) patients whereas those of other phosphatases such as PP2B are actually increased in the AD brain. PP2A activity negatively correlates to the level of tau phosphorylation at most phosphorylation sites in human brains. This indicates that PP2A, particularly PP2AB is the major tau phosphatase that regulates its phosphorylation at multiple sites in human brain. This implies that the abnormal hyperphosphorylation of tau is partially due to a downregulation of PP2A activity in AD brain and those agents that can act to boost the activity of PP2A, and in particular the ABC isoform of PP2A would have clinical utility in treating and or preventing development of neurodegenerative diseases.

PP2A, containing B' (B56, PR61 ) family subunits, participates in Wnt/_-catenin signaling, a signal transduction pathway necessary for vertebrate axis formation in early embryogenesis (Hsu, W., Zeng, L., and Costantini, F. (1999) J. Biol. Chem. 21 A, 3439-3445; Seeling, J. M., Miller, J. R., Gil, R., Moon, R. T., White, R., and Virshup, D. M. (1999) Science 283, 2089-20912, 3). B' subunits bind to cyclin G1 and G2, suggesting that PP2A holoenzymes containing these subunits are involved in cell cycle regulation (Okamoto, K., Kamibayashi, C, Serrano, M., Prives, C, Mumby, M. C, and Beach, D. (1996) MoI. Cell. Biol. 16, 6593-6602; Bennin, D. A., Arachchige Don, A. S., Brake, T., McKenzie, J. L., Rosenbaum, H., Ortiz, L., DePaoli-Roach, A. A., and Home, M. C. (2002) J. Biol. Chem. 277, 27449-27467; Okamoto, K., Li, H., Jensen, M. R., Zhang, T., Taya, Y., Thorgeirsson, S. S., and Prives, C. (2002) MoI. Cell 9, 761-771 ).

The B" subunit PR48 was first identified in a screen for proteins that interact with cdc6, a component of DNA prereplication complexes (Yan, Z., Fedorov, S. A.,

Mumby, M. C, and Williams, R. S. (2000) MoI. Cell.Biol. 20, 1021-1029), and may mediate the obligatory role of PP2A in the initiation of chromosomal DNA replication

(8). Another B" subunit, PR59, recently identified as an interaction partner for the retinoblastoma-related p107 protein (Voorhoeve, P. M., Hijmans, E. M., and Bernards, R. (1999) Oncogene 18, 515-524), could be important in the rapid dephosphorylation of p107 following DNA damage (Voorhoeve, P. M., Watson, R. J., Farlie, P. G., Bernards, R., and Lam, E. W. (1999) Oncogene 18, 679-688). PP2A holoenzymes containing B" family subunits may thus be specialized regulators of the G1/S cell cycle transition. Hence the roles of the PP2A and regulatory subunits (B, B' and B") are varied but specific.

Despite being the first PP2A regulatory subunits to be identified, roles of members of the B-type subunit family (PR55) are still largely enigmatic. In mammals, four genes (Bα-δ) code for 54-57-kDa proteins, which are additionally diversified by alternative splicing or promoter use. B (α-δ) are more than 80% identical at the amino acid level, with the greatest clustering of divergent residues at the N terminus. B-family PP2A subunits are predicted to adopt a seven-blade β-propeller fold similar to the β-subunits of heterotrimeric G proteins (Strack, S., Ruediger, R., Walter, G., Dagda, R. K., Barwacz, C. A., and Cribbs,J. T. (2002) J. Biol. Chem. 277, 20750-20755). Ba mediates dephosphorylation of vimentin intermediate filaments in fibroblasts (Turowski, P., Myles, T., Hemmings, B. A., Fernandez, A., and Lamb, N. J. (1999) MoI. Biol. Cell 10, 1997-2015) and has been shown to interact preferentially with microtubules and tau protein, possibly contributing to the regulation of microtubule stability by PP2A (Sontag, E., Nunbhakdi-Craig, V., Bloom, G. S., and Mumby, M. C. (1995) J. Cell Biol. 128, 1131-1 144; Sontag, E., Nunbhakdi-Craig, V., Lee, G., Bloom, G. S., and Mumby, M. C. (1996) Neuron 17, 1201-1207). Whereas Ba and Bδ are widely expressed in different tissues, Bβ and Bv proteins are detectable only in brain which suggests that Bβ and Bv mediate specifically neuronal functions of PP2A. Bβ and By expression is differentially regulated during development and maturation of the rat brain; Bβ protein and mRNA levels are high in late embryonic brain and decrease modestly after birth. In contrast, Bβ expression increases sharply postnatally to plateau at 2 weeks of age (Mayer, R. E., Hendrix, P., Cron, P., Matthies, R., Stone, S. R., Goris, J., Merlevede, W., Hofsteenge, J., and Hemmings, B. A. (1991 ) Biochemistry 30, 3589-3597; Zolnierowicz, S., Csortos, C, Bondor, J., Verin, A., Mumby, M. C, and DePaoli- Roach, A. A. (1994) Biochemistry 33, 1 1858-11867; Strack, S., Zaucha, J. A., Ebner, F. F., Colbran, R. J., and Wadzinski, B. E. (1998) J. Comp. Neurol. 392, 515-527) The method of screening enables compounds to be identified which can selectively regulate the PP2A especially the PP2AB form. Such compounds may ultimately be useful for the treatment of conditions associated with protein serine/threonine phosphatase activity, including those resulting from aberrant phosphorylation patterns such as in Alzheimer's disease. These compounds may act by restoring normal levels of specific forms of PP2A protein or PP2A enzymatic activity in cells specifically where specificity for a target protein is essential or restoring or reducing the phosphorylation levels of the PP2A substrate proteins. Selenate activates PP2A and reduces the amount of phosho-Tau in vivo. Surprisingly, overall Tau levels were also reduced. Therefore the compounds could be of use where a reduction in the levels of the phosphorylated from of the protein substrate are desirable (eg. Phospho-Tau), and potentially where a reduction in the protein substrate is required (eg. Tau). Alternatively, these compounds may act by increasing the level of protein serine/threonine phosphatase activity to a super-normal level, for example to offset an abnormally high level of protein kinase activity in the cell.

The advantage of identifying compounds that can activate specific forms of PP2A is that it would be useful clinically rather than using a form of PP2A which could dephosphorylate in an indiscriminate, pleiotropic manner.

Selenate also selectively activates complexes of PP2A that contain the PP2AB subunit complex. Particularly, sodium selenate boosts activity of the PP2A core dimer and shows substrate specificity for the PP2AB form of the PP2A.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described can be used in the practice or testing of the invention, preferred methods and materials are described.

Method of Screening

The methods employed will typically involve testing various compounds for their effect on the phosphatase activity of PP2A, particularly the PP2AB form. The compound identified will be specific for activating this form of PP2A to enable specificity of phosphatase activity. The compounds tested will be prepared for testing in a manner which enables the compound to react with the PP2A, particularly PP2AB so that a change of activity of the PP2A, particularly PP2AB may be measured. For instance, if the compound is water soluble, the compound will be dissolved in water to provide a range of various concentrations (including a control) to test the effect of the compound on the activity of the PP2A and PP2AB. A person skilled in the art will be able to determine suitable ways to prepare the sample for testing dependent upon the properties of the compound.

The compounds will be exposed to the samples under conditions suitable to allow for the compound to react and provide an effect on the PP2A in general and preferably PP2AB. Again, a person skilled in the art will be able to determine a suitable condition, in particular to measure the phosphatase activity of the PP2A and PP2AB. Conditions may include temperature and period of reaction.

The activity of the PP2A, especially the PP2AB form may be determined once or periodically over a period of time.

Any parameter which enables the determination of the phosphatase activity of PP2A or PP2AB can be used to ascertain whether the compound has any effect on the activity of the PP2A or the PP2AB.

The screening may be conducted on the compounds singularly or in combination so that single compounds or combinations of compounds can be tested for their effect on PP2A or more preferably PP2AB.

Without being limited by this description, the method of screening may be conducted simply by exposing a sample to a compound for testing to ascertain whether the compound affect firstly, phosphatase activity and then whether that phosphatase activity is attributed to the specific form of the PP2A being activated. Preferably, the PP2AB form will be activated and not the PP2AB' or the PP2AB".

Samples

The samples used for screening the compounds may be any sample that contains the PP2AB complex. It is through this complex that the applicants have identified a compound that can selectively activate this complex at the exclusion of others and hence provide specificity of this specific form of the PP2A.

Samples such as cells, cell lysates, primary cell cultures, tissues, tissue slices, tissue extracts, organotypic cultures nucleic acid, proteins such as enzymes and peptides can be used. In particular, it is desired to use cells and cell lysates that have been exposed to the compounds for testing the effect of the compound on the PP2A complex.

The samples may be obtained from cell cultures or whole animals that have been exposed to the compounds to be tested. If whole animals are used, any tissue from that animal may be analysed for an effect of the compound on the activity of the PP2A, specifically the PP2AB. For instance nerve tissue containing tau protein will be particularly susceptible to the activity of the PP2AB form of PP2A. This will be a useful tissue to monitor whether the compound affects the activity of the PP2AB but also provides additional in vivo data regarding the availability of the compound to the tissues in need.

Where proteins are tested with the compounds, upstream or downstream enzyme effects may be used to determine a change in phasphatase activity. For instance, the effect of the activity of PP2AB can be measured or monitored by the activity of proteins and enzymes affected downstream such as the GSK 3β, protein kinase Akt and the tau protein.

One type of abnormal tau protein is hyperphosphorylated tau protein. Tau protein is known to be phosphorylated at a number of phosphorylation sites by glycogen synthase kinase 3-beta (GSK3-beta) in vivo, including the Alzheimer's disease specific Ser396 residue. In turn, GSK3P is known to be phosphorylated by the protein kinase Akt and the activity of Akt is known to be attenuated by the protein phosphatase PP2A.

In cell cultures, these samples can be processed by methods available to the skilled addressee to release the PP2AB form for further analysis and determination of the phosphatase activity. Alternatively, the activity of the PP2A or PP2AB can be determined in situ where a tissue sample is used, by using a detector molecule such as an antibody.

Differential Effect The term "differential effect" as used herein refers to a difference in activity that the compound imparts on the PP2A, especially PP2AB in the presence and the absence of the compound. In one embodiment the differential effect is determined by a measurement of phosphatase activity of the PP2AB form of PP2A in the presence or absence of the compound. However, other parameters that affect the phosphatase activity such as changes in nucleic acid or amino acid sequences may also be considered to be a differential effect. Providing a comparison can be made between the presence and the absence of the compound, a differential effect can be measured. The purpose is to determine whether the compound has an effect on the activity of PP2AB. The differential effect may be activation or a deactivation of the PP2AB form of PP2A. Hence, the phosphatase activity may be regulated by activation or deactivation of the PP2A phosphatase activity.

The differential effect may also incorporate identifying whether the changed phosphatase activity is due to changed phosphatase activity in PP2AB or another form of PP2A such as PP2AB' or PP2AB". Hence it may enable determination of other forms of PP2A which can be selectively regulated.

Measuring a Differential Effect

Where the phosphatase activity is determined it may be determined directly or indirectly. Direct determination of the phosphatase activity may involve measuring the activity of the PP2A in general or PP2AB specifically directly in an assay which measures dephosphorylation of a phosphopeptide. Alternatively, the activity may be measured indirectly by measuring the effect of the PP2A or PP2AB on another protein such as tau protein via other downstream proteins such as protein kinase Akt and glycogen synthase kinase 3β (GSK3β).

The compound's effect may be measured as far downstream as in an in vivo model which tests the effect of reversing neurodegenerative diseases. For instance the compound's effect can be measured by considering the effect on behaviour and tau brain pathology of transgenic mice overexpressing human Tau 441 (TMHT). The progression of the behaviour may be measured by tests such as Open Field (OF) test, the Rota Rod (RR) test and the Nose Poke curiosity and activity test to evaluate curiosity behaviour.

In another embodiment, the invention may include a step of predetermining an effect of the compound to be screened on PP2A activity in general. Therefore, prior to determining the differential effect, a differential effect of the compound on phosphatase activity of a PP2A in general is determined to gauge whether the compound will affect phosphatase activity in general. If there is no differential effect in the presence and absence of the compound, it is unlikely that the compound will have any differential effect of the PP2AB form. Once a differential effect on the PP2A in general is determined, a further test to determine the effect on PP2AB can be conducted. This test will serve multiple purposes one of which is to determine whether the compound has a specific target for the PP2AB or whether the activity is due to the compound targeting another PP2A form such as PP2AB' or PP2AB". This pre-determination step will also provide an initial screening for those compounds that will affect the PP2A activity. This will provide a base level to further determine whether the effect of the compound is enhancing the PP2A activity or not via a PP2AB subunit complex.

The differential effect may be determined as an increase or decrease in the phosphatase activity of the PP2AB compared to a control in the absence of the compound.

Compounds that selectively regulate phosphatase activity of PP2A can be identified preferably by their ability to boost the activity of the PP2A specifically via the PP2AB subunit complex when compared to the activity of the PP2A containing the B subunit in the absence of the compound. Hence the method can select for those compounds that have a specific activity for the phosphatase.

Candidate Compounds

As described above the compounds may be tested alone or in combination. Once determined, the compounds may be used to develop compositions comprising one or more of the compounds identified.

Pharmaceutical Compositions and Methods of Treatment

Once a compound has been identified as herein described, the compound can be administered to a patient to treat a condition associated with the activity of a protein serine/threonine phosphatase, including those associated with an aberrant phosphatase activity. As discussed supra these conditions may include cancers, neurological disorders, angiogenic disorders, non-tumor diseases or disorders associated with aberrant angiogenesis.

For neurodegenerative conditions the present invention is predicated in part on the determination that selected compounds or a pharmaceutically acceptable salt thereof, is effective in enhancing the activity of PP2AB which in turn may result in a reduction in phosphorylation of tau protein by GSK3P and/or an increase in the rate of dephosphorylation of tau protein. The methods of the invention generally comprise exposing PP2AB present in neurons or glial cells to a PP2A activity enhancing amount of an identified compound or a pharmaceutically acceptable salt thereof. Suitably, the PP2A activity enhancing amount of an identified compound is a nutritional or supranutritional amount of an identified compound or a pharmaceutically acceptable salt thereof. In some embodiments, the amount of an identified compound or a pharmaceutically acceptable salt thereof, is from about 0.015 mg/kg to about 20 mg/kg, usually from about 0.1 mg/kg to 14 mg/kg, 0.07 mg/kg to 6.5 mg/kg or 0.15 mg/kg to 5 mg/kg per day, for example, 0.07 mg/kg to 2 mg/kg per day.

The compound identified in the present invention can be used effectively to treat or prevent neurodegenerative diseases. Neurodegenerative diseases include neurodegenerative movement disorders and neurodegenerative diseases associated with memory loss and include tauopathies and α-synucleopathies. Illustrative examples of neurodegenerative diseases include presenile dementia, senile dementia, Alzheimer's disease, Parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy (PSP), Pick's disease, primary progessive aphasia, frontotemporal dementia, corticobasal dementia, Parkinson's disease, Parkinson's disease with dementia, dementia with Lewy bodies, Down's syndrome, multiple system atrophy, amyotrophic lateral sclerosis (ALS) and Hallemorden-Spatz syndrome. In preferred embodiments, the invention is suitable for treating or preventing tauopathies. , In other embodiments, the invention is suitable for treating or preventing a-synucleopathy, especially Parkinson's disease. Suitably, the effective amount of an identified compound or a pharmaceutically acceptable salt thereof is a nutritional or supranutrional amount of an identified compound. In some embodiments, the amount of an identified compound or a pharmaceutically acceptable salt thereof, is from about 0.015 mg/kg to about 20 mg/kg, usually from about 0.1 mg/kg to 14 mg/kg or 0.07 mg/kg to 6.5 mg/kg or 0.15 mg/kg to 5 mg/kg per day, for example, 0.07 mg/kg to 2 mg/kg per day.

Where the condition is cancer, the cancer may be selected from the group consisting of brain, lung, liver, spleen, kidney, lymph node, small intestine, pancreas, blood cells, colon, stomach, breast, endometrium, prostate, testicle, ovary, skin, head and neck, esophagus, bone marrow and blood cancer. In more specific embodiments, the cancer is a lung cancer, colon cancer, breast cancer or cervical cancer.

Hyperproliferative disorders refers to excess cell proliferation, relative to that occurring with the same type of cell in the general population and/or the same type of cell obtained from a patient at an earlier time. The term denotes malignant as well as non-malignant cell populations. Such disorders have an excess cell proliferation of one or more subsets of cells, which often appear to differ from the surrounding tissue both morphologically and genotypically. The excess cell proliferation can be determined by reference to the general population and/or by reference to a particular patient, e.g. at an earlier point in the patient's life. Hyperproliferative cell disorders can occur in different types of animals and in humans, and produce different physical manifestations depending upon the affected cells. Hyperprol iterative cell disorders include cancers; blood vessel proliferative disorders such as restenosis, atherosclerosis, in-stent stenosis, vascular graft restenosis, etc.; fibrotic disorders; inflammatory disorders, e.g. arthritis, etc.; endometriosis; benign growth disorders such as prostate enlargement and lipomas; and autoimmune disorders. Cancers of particular interest include carcinomas, e.g. colon, prostate, breast, melanoma, ductal, endometrial, stomach, dysplastic oral mucosa, invasive oral cancer, non-small cell lung carcinoma, transitional and squamous cell urinary carcinoma, etc.; neurological malignancies, e.g. neuroblastoma, gliomas, etc.; hematological malignancies, e.g. childhood acute leukaemia, non-Hodgkin's lymphomas, chronic lymphocytic leukaemia, malignant cutaneous T-cells, mycosis fungoides, non-MF cutaneous T-cell lymphoma, lymphomatoid papulosis, T-cell rich cutaneous lymphoid hyperplasia, bullous pemphigoid, discoid lupus erythematosus, lichen planus, etc.; sarcomas, melanomas, adenomas; benign lesions such as papillomas, and the like, uterine, testicular and ovarian carcinomas, endometriosis, squamous and glandular epithelial carcinomas of the cervix. Other hyperprol iferative disorders that may be associated with altered activity of phosphorylation modifying enzyme(s) include a variety of conditions where there is proliferation and/or migration of smooth muscle cells, and/or inflammatory cells into the intimal layer of a vessel, resulting in restricted blood flow through that vessel, i.e. neointimal occlusive lesions. Occlusive vascular conditions of interest include atherosclerosis, graft coronary vascular disease after transplantation, vein graft stenosis, peri-anastomatic prosthetic graft stenosis, restenosis after angioplasty or stent placement, and the like. Other disorders and conditions of interest relate to epidermal hyperprol iferation, tissue remodelling and repair. For example, the chronic skin inflammation of psoriasis is associated with hyperplastic epidermal keratin ocytes.

The methods of the present invention are suitable for treating an individual who has been diagnosed with a neurodegenerative disease, who is suspected of having a neurodegenerative disease, or who is known to be susceptible and who is considered likely to develop a neurodegenerative disease. In some embodiments of the above methods, the neurodegenerative disease is a tauopathy, and the treatment optionally further comprises administration of another agent suitable for treating a taupathy as described above.

In other embodiments of the above methods, the neurodegenerative disease is an a- synucleopathy, especially Parkinson's disease and the treatment optionally further comprises administration of another agent suitable for treating an a-synucleopathy as described above.

Exemplary subjects for treatment with the methods of the invention are vertebrates, especially mammals. In certain embodiments, the subject is selected from the group consisting of humans, sheep, cattle, horses, bovine, pigs, dogs and cats. A preferred subject is a human.

It is to be understood that various other modifications and/or alterations may be made without departing from the spirit of the present invention as outlined herein. For instance, the skilled person would know that the scale of the features described herein may be altered without departing from the spirit of present the invention.

The present invention will now be further described by reference to the following non- limiting Examples.

EXAMPLES

EXAMPLE 1 : Identification of Compounds.

A. Screening For Candidate Compounds

This example describes a method of screening for candidate compounds by identifying simple substituents around a selenate and thioselenate core. Initially, QM calculation of representative species is performed using Jaguar to optimize the geometry and charge distribution of the various species, such as those depicted in

Figure 3 {e.g., selenates, thioselenates, selenate esters, etc.). The OPLS2001 force field is then parameterized for these compounds using the calculated properties. Once this has been performed, a virtual screen of the substituted selenates and thioselenates is performed using the Glide software. Refinement of the docking results is undertaken by using QMDock and Liaison calculations. All software mentioned is part of the Schrodinger LLC suite of software.

B. Docking Calculations

The docking calculations were performed as follows using Glide, v 4.5 (Schrodinger, LLC):

Energy grids were calculated around each site. Proposed compounds were positioned in each grid and a score was calculated that indicates whether the position of the compound is favourable in energy. For compounds that have many conformations, Glide performs a preliminary conformational analysis and each conformation was posed on the energy grid. Highly favourable geometries were further scored using a different equation to estimate whether the binding energy will be favourable. Additionally, selenate compounds were parameterized and for these calculations. Sulfate was docked as parameterized in the software.

C. Modeling Interactions of Selenate and Selenate Derivatives with PP2A

Two attractive positions for selenate were identified in the active site of the catalytic subunit of PP2A. Quantum mechanical calculations were used to parameterize selenate for use with docking programs. Selenate parameters for MM calculations have been validated in small systems.

D. Binding Energy Contributions of Sulfate and Selenate to the PP2A Catalytic Subunit

(i) Protein preparation:

• Selenate binding in the vicinity of the acid group of okadaic acid bound to the PP2A active site (pdb code 2IE4)

• Changed metal ions to Mg2+ (gives similar results in calculation to Mn2+ but is easier to treat quantum mechanically, molecular mechanics also similar).

• Acidic and basic side chain protonation states assigned by pprep (Schrodinger, LLC, New York, NY, impact v 4.5.108). • Changed proton assignment of HIS which is ligated to the Mg2+. • Assigned additional waters to fill coordination shell of Mg2+ as can be observed in the various crystal structures.

• Minimized protein with impact (Schrodinger, LLC, New York, NY, impact v 4.5.108).

• Second minimization with impact following placement of selenate by docking with glide (Schrodinger, LLC, New York, NY, Glide v4.5 .108).

(H) Binding Calculations:

• QM Caguar; Schrodinger, LLC, New York, NY, jaguar v1.165.2.2): DFT/B3LYP with lacvp^* basis set • QM/MM (qsite; Schrodinger, LLC, New York, NY, qsite v1.6.4.1 ): QM region: DFT/B3LYP with lacvp^* basis set; MM region: impact with OPLS2001 force field parameterized for selenate

• MM (impact; Schrodinger, LLC, New York, NY, impact v 4.5.108): OPLS2001 force field parameterized for selenate.

E. Binding Energy Contributions of Sulfate and Selenate to PP2A Catalytic Subunit

Binding energy terms (in kcal/mol) were collected with QM/MM and MM: • QM/MM: Binding energy terms following optimization:

• MM: Binding energy terms using optimized QM/MM structure:

The results show that binding energy differences between sulfate and selenate (in kcal/mol) of -49.3 (QM/MM) and -38.5 (MM). F. Binding affinity of selenate/Sulfate using docking algorithms:

• Regular docking with Glide, XP score and OPLS2001 force field with parameterized selenate.

• QM docking using qpld with XP scoring. Charges were calculated quantum mechanically for a free ion in water (implicit solvent model).

Table 3:

G. List of Binding Motifs Investigated and their Locations

Tables 3:

EXAMPLE 2: Treatment of Advanced Non-Small Cell Lung Cancer (NSCLC).

A compound of the present invention is prepared as a pharmaceutically acceptable salt and formulated as a sterile composition for IV infusion using phosphate buffered saline.

The patient has stage NIB or IV disease (histologically proven), adequate hematologic, hepatic and renal function, and ECOG performance status 0, 1 or 2. Baseline tests are performed: CBC & diff, platelets, serum creatinine, LFT's, chest X- ray, camera nuclear renogram for GFR (if available). Before and after each treatment, the following tests are performed: CBC & diff, serum creatinine, any initially elevated tumor marker, LFT's (if clinically indicated).

The patient is infused with the equivalent of 1 mg/kg of compound over four hours by IV infusion. Treatment is repeated daily until tumour growth is slowed, halted, or reversed, or until side effects (if any) become intolerable.

EXAMPLE 3: Treatment of Colon Cancer.

The patient has resected Stage III or high risk Stage Il colon cancer, adequate hematologic, hepatic and renal function, and ECOG performance status 0, 1 or 2.

Baseline tests are performed: CBC and differential, liver function tests and creatinine. Before and after each treatment, the following tests are performed: CBC and differential creatinine. If clinically indicated: liver function tests, BUN.

EXAMPLE 4: Treatment of Alzheimer's Disease.

A compound of the present invention is prepared as a pharmaceutically acceptable salt and formulated as a solid dosage form for oral administration.

The patient has diagnosed Alzheimer's disease (diagnosed by NINCDS-ADRDA and DSM-IV criteria, Mini-Mental State Examination (MMSE) =10 and =26, and the Global Deterioration Scale (GDS)).

The patient is administered with the equivalent of 1 mg/kg by single daily dose of compound. Treatment is repeated daily until decline in congnitive performance is slowed, halted, or reversed, or until side effects (if any) become intolerable. The ability of the compound to improve cognitive performance is assessed using the cognitive subscale of the Alzheimer's Disease Assessment Scale (ADAS-cog), a multi-item instrument that has been extensively validated in longitudinal cohorts of Alzheimer's disease patients. The ADAS-cog examines selected aspects of cognitive performance including elements of memory, orientation, attention, reasoning, language and praxis. The ADAS-cog scoring range is from 0 to 70, with higher scores indicating greater cognitive impairment.

EXAMPLE S: Treatment of Psoriasis.

A compound of the present invention is prepared as a pharmaceutically acceptable salt and formulated as a gel for topical administration. The compound is present in the gel at a concentration of 1 % w/w.

The patient has active, plaque psoriasis involving greater than or equal to 10% of the body surface area, and a minimum PASI score of 10.

The gel is liberally applied to affected skin three times per day, until symptoms decrease to an acceptable level, or until the side effects (if any) are intolerable.

Future patent applications may be filed on the basis of or claiming priority from the present application. It is to be understood that the following provisional claims are provided by way of example only, and are not intended to limit the scope of what may be claimed in any such future application. Features may be added to or omitted from the provisional claims at a later date so as to further define or re-define the invention or inventions.

EXAMPLE 6: In Vitro Screening of Compounds.

Primary rat neurons cultured with screen compounds (ie sodium selenate concentration range 5 -100μM for 1 -6hrs) and then PP2A B subunit immunoprecipated and activity measured with Malachite green assay.

(i) Primary neuron cell culture and isolation of neuronal cell lysates

To culture primary neurons, 15 fresh brains of 1-day postnatal rat pups were cut into small pieces and then transferred into a 50-ml Falcon tube containing 30 ml of PBS

(25 mM Na₂HPO₄ (pH 7.4), 137 mM NaCI). After adding 3 ml of trypsin (2.5%), the tube was incubated for 15 min at 37 ⁰C with occasional mixing by tube inversion. To the incubated mixture, 3 ml of fetal bovine serum (HyClone, Logan, UT) followed by 4 ml of DNase I (1 mg/ml) were added. The tube was subjected to five or six inversions followed by gentle trituration using a 25-ml glass pipette until the mixture became homogeneous. The mixture was filtered twice through a nylon membrane. The neurons in the filtrate were pelleted by centrifugation for 10 min at 10 ⁰C. The pellet was washed twice with PBS and then dispersed in 45 ml of culture medium (minimum essential medium (Invitrogen) supplemented with 30 mM glucose, 2 mM glutamine, 1 mM pyruvate, and 10% fetal bovine serum). Neurons in the dispersed solution were plated at 3 x 10⁶ cells/mm in 35-mm poly-D-lysine-coated dishes and placed in aCO₂ incubator maintained at 37 ⁰C. The culture medium was replaced with fresh culture medium supplemented with 1 mM fluorodeoxyuridine (Sigma) on the second and the third days. On the fourth day, the culture medium was again replaced with fresh culture medium without fluorodeoxyuridine. On the seventh day, neurons were scraped and suspended in 0.5 ml of lysis buffer (20 mM imidazole-HCI, 2 mM EDTA, 2 mM EGTA, pH 7.0 with aprotinin, benzamidine and AEBSF (all from Sigma-Aldrich, St Louis, MO, USA)). The suspension was incubated on ice with occasional shaking for 30 min and centrifuged, and the supernatant was used for a immunoprecipitation assay.

(ii) PP2A assay

PP2A immunoprecipitation and activity were determined using a non-radioactive kit according to the manufacturer's instructions (cat. no. 17-127, Upstate Biotechnologies, Lake Placid, NY, USA). Primary rat neuronal cell lysates were prepared in 20 mM imidazole-HCI, 2 mM EDTA, 2 mM EGTA, pH 7.0 with aprotinin, benzamidine and AEBSF (all from Sigma-Aldrich, St Louis, MO, USA). Protein concentrations were determined using BCA and 0.5-1.0 mg protein was immunoprecipitated using an anti- PP2A B subunit antibody (Upstate, Lake Placid, NY, USA) and protein A-Sepharose beads (Zymed Laboratories, South San Francisco, CA, USA). Equivalent immunoprecipitation of PP2A from all samples was confirmed by immunoblot. Immunoprecipitated PP2A was then tested for activity in a 10-minute reaction at 37°C, in which phosphopeptide (K-R-pT-l-R-R) dephosphorylation was assayed spectrophotometrically at 650 nm using Malachite Green. PP2A activity was determined for all samples relative to a phosphate standard curve with activity expressed as pmol incorporated phosphate/minute/μg protein. EXAMPLE 7: Treatment of Mice with Screened Compounds.

Mice treated with screen compounds ( ie sodium selenate in drinking water, 7 days at 1.2 mg/100 ml drinking water) and then mice sacrificied and brain extracts prepared and brain PP2A activity of PP2A containing the B subunit form immunoprecipitated and activity checked.

(i) lmmunoprecipitation of PP2A B subunit from mouse brain extracts

For immunoprecipitation all steps were carried out at 4°C. Adult mice striata were collected, weighed and homogenized in 5 volumes of ice-cold co-IP buffer, which contained 50 mM Tris pH 7.4, 100 mM NaCI, 5 mM EDTA, 0.3% Triton X-100, 10% glycerol plus aprotinin, leupeptin, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), β-glycerophosphate, and dithiothreitol to inhibit protease and phosphatase activities. Supernatants were collected after centrifugation at 17,000 g (Sorvall RC5B, Kendro Laboratory Products, Newtown, CT, USA). A control aliquot of each supernatant was separated and frozen prior to IP for total protein determinations. Samples were pre- cleared for 1 hour with 10 μl 1 % BSA plus 25 μl each of protein A- and protein G- Sepharose beads (Zymed Laboratories, S. San Francisco, CA, USA), lmmunoprecipitating antibodies (5 μg, PP2A B subunit, Upstate) were coupled to Sieze™ X beads according to the manufacturer's instructions (Pierce, Rockford, IL, USA). Equal aliquots of homogenate (5.0 mg/ml total protein) were incubated with antibodies or pre-absorbed antibodies. Immune complexes were eluted, separated on 10 or 15% Tris-glycine SDS-PAGE gels, transferred to nitrocellulose, reacted with the same primary antibodies described above, and visualized by chemiluminescence.

(ii) PP2A assay

PP2A immunoprecipitation and activity were determined using a non-radioactive kit according to the manufacturer's instructions (cat. no. 17-127, Upstate Biotechnologies, Lake Placid, NY, USA). Mouse brain lysates were prepared in 20 mM imidazole-HCI, 2 mM EDTA, 2 mM EGTA, pH 7.0 with aprotinin, benzamidine and AEBSF (all from Sigma-Aldrich, St Louis, MO, USA). Protein concentrations were determined using BCA and 0.5-1.0 mg protein was immunoprecipitated using an anti-PP2A B subunit antibody (Upstate, Lake Placid, NY, USA) and protein A-Sepharose beads (Zymed Laboratories, South San Francisco, CA, USA). Equivalent immunoprecipitation of PP2A from all samples was confirmed by immunoblot. Immunoprecipitated PP2A was then tested for activity in a 10-minute reaction at 37°C, in which phosphopeptide (K-R- pT-l-R-R) dephosphorylation was assayed spectrophotometrically at 650 nm using Malachite Green. PP2A activity was determined for all samples relative to a phosphate standard curve with activity expressed as pmol incorporated phosphate/minute/μg protein.

EXAMPLE 8: Effect of screened compounds on Tau protein. Tau protein is immunoprecipated from mouse brain extracts from mice treated with test compounds (ie sodium selenate in drinking water, 7 days at 1.2 mg/100 ml drinking water) and then mice sacrificied and brain extracts prepared and brain PP2A activity associated with brain tau coimmunoprecipitated and activity checked.

(i) Co-immunoprecipitation of tau and PP2A

For coimmunoprecipitation all steps were carried out at 4°C. Adult mice striata were collected, weighed and homogenized in 5 volumes of ice-cold co-IP buffer, which contained 50 mM Tris pH 7.4, 100 mM NaCI, 5 mM EDTA, 0.3% Triton X-100, 10% glycerol plus aprotinin, leupeptin, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), β-glycerophosphate, and dithiothreitol to inhibit protease and phosphatase activities. Supernatants were collected after centrifugation at 17,000 g (Sorvall RC5B, Kendro Laboratory Products, Newtown, CT, USA). A control aliquot of each supernatant was separated and frozen prior to colP for total protein determinations. Samples were pre- cleared for 1 hour with 10 μl 1 % BSA plus 25 μl each of protein A- and protein G- Sepharose beads (Zymed Laboratories, S. San Francisco, CA, USA), lmmunoprecipitating antibodies (5 μg,anti-tau Cell signaling technologies) were coupled to Sieze™ X beads according to the manufacturer's instructions (Pierce, Rockford, IL, USA). Equal aliquots of homogenate (5.0 mg/ml total protein) were incubated with antibodies or pre-absorbed antibodies. Immune complexes were eluted, separated on 10 or 15% Tris-glycine SDS-PAGE gels, transferred to nitrocellulose, reacted with the same primary antibodies described above, and visualized by chemiluminescence.

(ii) PP2A assay

PP2A immunoprecipitation and activity were determined using a non-radioactive kit according to the manufacturer's instructions (cat. no. 17-127, Upstate Biotechnologies, Lake Placid, NY, USA). Mouse brain lysates were prepared in 20 mM imidazole-HCI, 2 mM EDTA, 2 mM EGTA, pH 7.0 with aprotinin, benzamidine and AEBSF (all from Sigma-Aldrich, St Louis, MO, USA). Protein concentrations were determined using BCA and 0.5-1.0 mg protein was immunoprecipitated using an anti-tau antibody (Cell Signaling Technologies) and protein A-Sepharose beads (Zymed Laboratories, South San Francisco, CA, USA). Equivalent immunoprecipitation of PP2A from all samples was confirmed by immunoblot. Immunoprecipitated PP2A was then tested for activity in a 10-minute reaction at 37°C, in which phosphopeptide (K-R-pT-l-R-R) dephosphorylation was assayed spectrophotometrically at 650 nm using Malachite Green. PP2A activity was determined for all samples relative to a phosphate standard curve with activity expressed as pmol incorporated phosphate/minute/μg protein.

NEW AMRl DATA

EXAMPLE 9: Decomposition of the Interaction Energies

The residue contribution to the interaction energies at sites 2/15, 28/40, and 10, 32 were investigated. Site 10 corresponds to the head piece of okadaic acid (as shown in pdbcode 2IE4) in proximity to the metal binding site. Site 2/15 corresponds to the tail piece of okadaic acid as shown in pdbcode 2IE4. Sites 28/40 and 32 are other small binding sites on PP2A with potential for binding of small ions such as SeO₄ ^2-.

(a) Site 10 Interaction Energies^* Referring to Figure 15 (structure at left) , there is shown three alternate docked locations of selenate in site 10 of optimized PP2A (pdbcode 2IE4). Referring to Figure 15 (structure at right) Below right there is shown an overlay of three alternate docked location with okadaic acid from pdbcode 2IE4. Occupation of site 10_B corresponds to the location of the acid head group of okadaic acid. Two arginine side chains (R89 and R214) are positioned ideally to stabilize small ions such as SeO₄ ^2-.

Site 10 Interaction Energies

Non bonded interaction energies (in kcal/mol) for amino acids close to SeO₄ ^" in binding sites 10_A, 10_B and 1-_C.

It will be noted that all three locations dominated by interactions with charged side chains of R89 and R214. Contributions from other amino acids result in only a slight shift in position. Residues H241 , H118 and Q242 are involved in stabilizing metal centers and are likely to contribute little to stabilization of SeO₄ ^2".

(b) Site 2/15 Interaction Energies

Figure 16 shows the location of selenate in site 2/15 of optimized PP2A (pdbcode 2IE4). This corresponds to the location of the tail piece of okadaic acid (yellow carbon atoms). One histidine side chain (H191 ) and a glutamine side chain (Q122) stabilize SeO₄ ^2". Side chains of E188 and R121 are involved in an ionic interaction which may make the contribution of these side chains to the binding energy less important than is implicated by individual interaction energies reported herein. Site 2/15 Interaction Energies

Non bonded interaction energies (in kcal/mol) for amino acids close to selenate in binding site 2/15.

(c) Site 28/40 Interaction Energies

Figure 17 shows docked location of selenate in site 28/40 of optimized PP2A (pdbcode 2IE4). SeO₄ ^2" is stabilized by hydrogen bond to backbone NH of H252 and D253 as well as interactions with charged side chain of H252 (dashed lines). Alternate rotameric orientation of Q222 would likely further stabilize selenate in this site.

Site 28/40 Interaction Energies

Non bonded interaction energies (in kcal/mol) for amino acids close to selenate in binding site 28/40. (d) Site 32 Interaction Energies

Figure 18 shows the docked location of selenate in site 32 of optimized PP2A

(pdbcode 2IE4). SeO42- stabilized by hydrogen bond to backbone NH of D290 (dashed line).

Site 32 Interaction Energies

Non bonded interaction energies (in kcal/mol) for amino acids close to selenate in binding site 32.

EXAMPLE 10: Characterization of Selenium Systems Selenium containing compounds were parameterized (see Figure 19). Quantum mechanical (QM) optimization (Jaguar, version 7.0, Schrodinger, LLC, New York, NY, 2007) of a selenite with a LAV2P^* basis set were used to obtain atomic charges from electrostatic potentials (in red) and bond distances (in grey). Van der Waals parameters for selenium were obtained from the literature (Zhao, L.; Cox, A.G.; Ruzicka, J.A.; Bhat, A.A.; Zhang, W.; Taylor, E.W. Proc. Natl. Acad. Sci. USA 2000, 97, 6356-6361 ).

Parameters were incorporated in the OPLS2001 force field for Impact (Impact, version 45213, Schrodinger, LLC, New York, NY, 2007) molecular mechanics, Macromodel interaction energies and Glide (Glide, version 45213, Schrodinger, LLC, New York, NY, 2007) molecular docking. All calculations for the regular docking are done using the extra precision glide mode (Friesner, R. A.; Murphy, R. B.; Repasky, M. P.; Frye, L. L.; Greenwood, J. R.; Halgren,T. A.; Sanschagrin, P. C; Mainz, D. T., "Extra Precision Glide: Docking and Scoring Incorporating a Model of Hydrophobic Enclosure for Protein-Ligand Complexes", J. Med. Chem., 2006, 49, 6177-6196) EXAMPLE 11 : Docking Affinity Evaluation of Selenate and Selenite

Previous modeling studies indicated that docking scores were appropriate to evaluate the relative binding affinity of selenium containing compounds. Binding affinity of selenite was performed using docking algorithms:

• Regular docking with Glide, XP score and OPLS2001 force field with parameterized compounds.

• QM docking using qpld with XP scoring. Charges calculated quantum mechanically for a free ion in water (implicit solvent model).

Docking of selenate and selenite to the sites of high interest indicates the affinity of these compounds for PP2A are as follows: SeO₄ ^2" > SeO₃ ^2".

EXAMPLE 12: Characterization of Selenium Dioxide

Selenium dioxide was parameterized for use in molecular docking calculations:

Atom: Selenium (atomic no 34) MW 78.96

Quantum mechanical (QM) optimization (Jaguar, version 7.0, Schrodinger, LLC, New York, NY, 2007) of selenium dioxide with a LAV2P^* basis set was used to obtain atomic charges from electrostatic potentials (in red) and bond distances (in grey), as shown in Figure 20. Van der Waals parameters for selenium are obtained from the literature (Zhao, L.; Cox, A.G.; Ruzicka, J.A.; Bhat, A.A.; Zhang, W.; Taylor, E.W. Proc. Natl Acad. Sci. USA 2000, 97, 6356-6361 ).

Parameters were incorporated in the OPLS2001 force field for Impact (Impact, version 45213, Schrodinger, LLC, New York, NY, 2007 molecular mechanics) and Glide molecular docking (Glide, version 45213, Schrodinger, LLC, New York, NY, 2007).

Bonds:

Angles:

The dihedral parameters for dimethyl selenone were set to be the same as for sulfones. EXAMPLE 13: Docking Affinity Evaluation of Selenium Dioxide

Previous modeling studies indicated that docking scores were appropriate to evaluate the relative binding affinity of selenium containing compounds. Binding affinity of selenium dioxide performed using docking algorithms:

QM docking using qpld with XP scoring. Charges calculated quantum mechanically for a free ion in water (implicit solvent model). The overall observed negative interaction energies for selenium dioxide indicates it would also bind to the protein in similar locations to other selenium containing compounds.

EXAMPLE 14: Modulatory Activity of Selenium Species on Purified PP2A AC Core Dimer.

Phosphatase Assays: Purified human PP2Ac (Upstate) was diluted to 0.01 U/μl with the dilution buffer provided and stored in aliquots at -20⁰C.

Phosphopeptide Assays with Purified Phosphatases: 1 mg of synthetic phosphopeptides K-R-pT-l-R-R (Upstate #12-219) and R-R-A-pS-V-A (Upstate #12-

220) were dissolved in 1.10-1.285 ml of dH₂O to prepare a 1 mM solution, then aliquoted and stored at -20⁰C until use. 0.01-0.05 U of purified PP2A A-C dimer purified from human red blood cells, was mixed with 500 μM of phosphopeptide and incubated in the presence of various selenium species as indicated (all at 50μM) as indicated at 37oC on a heating block for 15 min. Each reaction was made up to a total volume of 25 μl with pNPP buffer (50 mM Tris-HCI, pH 7.0, 100 μM CaCI2). The enzyme reaction was terminated by adding 100 μl of malachite green solution

(0.034% malachite green in 10 mM ammonium molybdate, 1 N HCI, 3.4% ethanol,

0.01% Tween-20). Malachite green forms a stable green complex in the presence of molybdate and orthophosphate, allowing the amount of inorganic phosphate present to be measured. Absorbance was read in duplicate for each sample at 590 nm.

These results shown in Figure 13 demonstrate that sodium selenate, sodium selenite, and selenium dioxide are inducers of PP2A phosphatase activity. The results further suggest that there is some specificity in inducing PP2A phosphatase activity amongst different selenium species, and that the core structure of the selenium species rather than the presence of the selenium ion itself may be mediating this effect.

Claims

1. A compound capable of modulating protein serine/threonine phosphatase activity, the compound having the formula:

or

R¹ R², R³ and R⁴ are each independently selected from the group consisting of: =O, =S, OR⁵, SR⁵, NR⁵, H, optionally substituted C₁-C₁₂ alkyl, optionally substituted C₂-C₁₂ alkenyl, optionally substituted C₂-C₁₂ alkynyl, optionally substituted C₁-C₁₀ heteroalkyl, optionally substituted C₃-C₁₂ cycloalkyl, optionally substituted C₃-C₁₂ cycloalkenyl, optionally substituted C₂- C₁₂ heterocycloalkyl, optionally substituted C₂-C₁₂ heterocycloalkenyl, optionally substituted C₆-C₁₈ aryl, optionally substituted CrC₁₈ heteroaryl, and acyl;

each R⁵ is independently selected from the group consisting of: H, optionally substituted CrC₁₂ alkyl, optionally substituted C₂-C₁₂ alkenyl, optionally substituted C₂-C₁₂ alkynyl, optionally substituted C₁-C₁₀ heteroalkyl, optionally substituted C₃-C₁₂ cycloalkyl, optionally substituted C₃-C₁₂ cycloalkenyl, optionally substituted C₂-C₁₂ heterocycloalkyl, optionally substituted C₂-C₁₂ heterocycloalkenyl, optionally substituted C₆-C₁₈ aryl, optionally substituted CrC₁₈ heteroaryl, and acyl; m is an integer selected from 0 and 1 ,

n is an integer selected from 0 and 1 ,

p is an integer selected from 0 and 1 ,

q is an integer selected from 0 and 1 ,

the sum of m+n+p+q is an integer from 2 to 4,

or a pharmaceutically acceptable salt thereof.

2. A compound according to claim 1 wherein X is selenium.

3. A compound according to claim 1 or claim 2 wherein n is 1.

4. A compound according to claim 1 or claim 2 wherein n is 2.

5. A compound according to claim 1 wherein the compound is

or a pharmaceutically acceptable salt thereof.

6. A compound according to claim 1 wherein the compound is selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

7. A compound according to claim 1 wherein the compound is selected from the group consisting of

or a pharmaceutically acceptable salt thereof.

8. A compound according to any one of claims 1 to 7 wherein R¹ is selected from the group consisting of H, CH₃, CH₂CH₃, CH₂CH₂CH₃, CH(CH₃)₂, CH₂CH₂CH₂CH₃, C(CH₃)₃,and phenyl.

9. A compound according to any one of claims 1 to 8 wherein R² is selected from the group consisting of H, CH₃, CH₂CH₃, CH₂CH₂CH₃, CH(CH₃)₂, CH₂CH₂CH₂CH₃, C(CH₃)₃,and phenyl.

10. A compound according to any one of claims 1 to 9, wherein the compound is selected from the group consisting of SO₄ ^2", SeO₄ ^2", SeO₂(CH₃)₂, SeO₃CH₃ ^", SeO₂CH₃,

Se₂O₃ ^2", SeO₃SH^", or a pharmaceutically acceptable salt thereof.

11. A compound according to any one of claims 1 to 11 , wherein the compound is capable of upregulating the activity of a protein serine/threonine protein phosphatase.

12. A compound according to claim 11 wherein the protein serine/threonine phosphatase is selected from the group consisting of PP1 α, PP1 β, PP1γ1 , PP1γ2, PP2A, PP2B , PP2C, PP4, PP5 and PP6.

13. A compound according to claim 11 wherein the protein serine/threonine phosphatase is PP2A.

14. A composition comprising a compound according to any one of claims 1 to 13 in combination with a pharmaceutically acceptable carrier.

15. A method for upregulating the activity of a serine/threonine protein phosphatase, the method comprising the step of exposing the cell to an effective amount of a compound according to any one of claims 1 to 13, or a composition according to claim 14.

16. A method according to claim 15 wherein the protein serine/threonine phosphatase is selected from the group consisting of PP1 α, PP1 β, PP1γ1 , PP1γ2, PP2A, PP2B, PP2C, PP4, PP5 and PP6.

17. A method according to claim 16, wherein the protein serine/threonine phosphatase is PP2A.

18. A method for treating or preventing a condition associated with the activity of a serine/threonine protein phosphatase, the method comprising administering to a subject in need thereof a therapeutically effective amount of a composition according to claim 14.

19. A method according to claim 18 wherein the protein serine/threonine phosphatase is selected from the group consisting of PP1 α, PP1 β, PP1γ1 , PP1γ2, PP2A, PP2B, PP2C, PP4, PP5 and PP6.

20. A method according to claim 18, wherein the protein serine/threonine phosphatase is PP2A.

21. A method according to any one of claims 15 to 20 wherein the condition associated with the activity of a serine/threonine protein phosphatase is a hyperproliferative disorder.

22. A method according to claim 21 wherein the hyperproliferative disorder is cancer.

23. A method according to claim 22 wherein the cancer is selected from the group consisting of brain, lung, liver, spleen, kidney, lymph node, small intestine, pancreas, blood cells, colon, stomach, breast, endometrium, prostate, testicle, ovary, skin, head and neck, esophagus, bone marrow and blood cancer; carcinomas (comprising colon, prostate, breast, melanoma, ductal, endometrial, stomach, dysplastic oral mucosa, invasive oral cancer, non-small cell lung carcinoma, transitional and squamous cell, urinary carcinoma); neurological malignancies (comprising neuroblastoma, gliomas); hematological malignancies, (comprising childhood acute leukaemia, non-Hodgkin's lymphomas, chronic lymphocytic leukaemia, malignant cutaneous T-cells, mycosis fungoides, non-MF cutaneous T-cell lymphoma, lymphomatoid papulosis, T-cell rich cutaneous lymphoid hyperplasia, bullous pemphigoid, discoid lupus erythematosus, lichen planus; sarcomas, melanomas, adenomas; benign lesions such as papillomas, and the like, uterine, testicular and ovarian carcinomas, endometriosis, squamous and glandular epithelial carcinomas of the cervix.

24. A method according to claim 23 wherein the hyperproliferative disorder is a vascular hyperproliferative disorder

25. A method according to claim 24 wherein the vascular hyperproliferative disorder is selected from the group consisting of restenosis , in-stent stenosis, and vascular graft restenosis, neointimal occlusive lesions, atherosclerosis, graft coronary vascular disease after transplantation, vein graft stenosis, peri-anastomatic prosthetic graft stenosis, restenosis after angioplasty or stent placement.

26. A method according to claim 23 wherein the hyperproliferative disorder is selected from the group consisting of a fibrotic disorder, an inflammatory disorder (e.g. arthritis), endometriosis; psoriasis, a benign growth disorder (comprising prostate enlargement and lipoma); and an autoimmune disorder (comprising scleroderma, systemic lupus erythematosus, Sjogren's syndrome, atopic dermatitis, asthma, and allergy).

27. A method according to any one of claims 18 to 26 wherein the condition associated with the activity of a serine/threonine protein phosphatase is a neurodegenerative condition.

28. A method according to claim 27 wherein the neurodegenerative condition is a tauopathy.

29. A method according to claim 27 or claim 28 wherein the neurodegenerative condition is selected from the group consisting of Creutzfeldt-Jakob disease,

Huntington's disease, stroke, cerebral ischaemia, dementia associated with stroke or cerebral ischaemia, dementia associated with HIV, disorders associated with excitotoxicity, epilepsy, seizures, schizophrenia, bipolar disorder, depression, mood disorder, multiple sclerosis, acute brain trauma (comprising severe traumatic brain injury) and motor neurone disease.

30. A method according to claim 29 wherein the neurodegenerative condition is selected from the group consisting of presenile dementia, senile dementia, Alzheimer's disease, Parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy (PSP), Pick's disease, primary progressive aphasia, frontotemporal dementia, corticobasal dementia, Parkinson's disease, dementia with Lewy bodies, Down's syndrome, multiple system atrophy, amyotrophic lateral sclerosis (ALS), multiple sclerosis, and Hallervorden-Spatz syndrome.

31. A method according to any one of claims 18 to 26 wherein the condition associated with the activity of a serine/threonine protein phosphatase is a non-tumor disease or disorder associated with aberrant angiogenesis.

32. A method according to claim 31 wherein the non-tumor disease or disorder associated with aberrant angiogenesis is selected from the group consisting of a clinical condition characterised by excessive vascular endothelial cell proliferation, vascular permeability, edema or inflammation comprising macular degeneration, especially age-related macular degeneration comprising wet macular degeneration and dry macular degeneration; a retinopathy such as diabetic retinopathy, ischaemic retinal vein occlusion and retinopathy of prematurity; endometriosis, restenosis, psoriasis and rheumatoid arthritis, brain edema associated with injury, stroke or tumor, edema associated with inflammatory conditions such as psoriasis, and arthritis comprising rheumatoid arthritis, asthma, generalised edema associated with burns, ascites and pleural effusion, especially macular degeneration, diabetic retinopathy, endometriosis and restenosis.

33. Use of a compound according to any one of claims 1 to 13 in the manufacture of a medicament for the treatment or prevention of a condition associated with the activity of a serine/threonine protein phosphatase.

34. Use according to claim 33 wherein the protein serine/threonine phosphatase is selected from the group consisting of PP1 α, PP1 β, PP1γ1 , PP1γ2, PP2A, PP2B, PP2C, PP4, PP5 and PP6.

35. Use according to claim 33, wherein the protein serine/threonine phosphatase is PP2A.

36. Use according to any one of claims 33 to 35 wherein the condition associated with the activity of a serine/threonine protein phosphatase is a hyperproliferative disorder.

37. Use according to claim 36 wherein the hyperproliferative disorder is cancer.

38. Use according to claim 37 wherein the cancer is selected from the group consisting of brain, lung, liver, spleen, kidney, lymph node, small intestine, pancreas, blood cells, colon, stomach, breast, endometrium, prostate, testicle, ovary, skin, head and neck, esophagus, bone marrow and blood cancer; carcinomas (comprising colon, prostate, breast, melanoma, ductal, endometrial, stomach, dysplastic oral mucosa, invasive oral cancer, non-small cell lung carcinoma, transitional and squamous cell, urinary carcinoma); neurological malignancies (comprising neuroblastoma, gliomas).; hematological malignancies, (comprising childhood acute leukaemia, non-Hodgkin's lymphomas, chronic lymphocytic leukaemia, malignant cutaneous T-cells, mycosis fungoides, non-MF cutaneous T-cell lymphoma, lymphomatoid papulosis, T-cell rich cutaneous lymphoid hyperplasia, bullous pemphigoid, discoid lupus erythematosus, lichen planus; sarcomas, melanomas, adenomas; benign lesions such as papillomas, and the like, uterine, testicular and ovarian carcinomas, endometriosis, squamous and glandular epithelial carcinomas of the cervix.

39. Use according to claim 36 wherein the hyperproliferative disorder is a vascular hyperproliferative disorder

40. Use according to claim 39 wherein the vascular hyperproliferative disorder is selected from the group consisting of restenosis , in-stent stenosis, and vascular graft restenosis, neointimal occlusive lesions, atherosclerosis, graft coronary vascular disease after transplantation, vein graft stenosis, peri-anastomatic prosthetic graft stenosis, restenosis after angioplasty or stent placement.

41. Use according to claim 36 wherein the hyperproliferative disorder is selected from the group consisting of a fibrotic disorder, an inflammatory disorder (comprising arthritis), endometriosis; psoriasis, a benign growth disorder (comprising prostate enlargement and lipoma); and an autoimmune disorder (comprising scleroderma, systemic lupus erythematosus, Sjogren's syndrome, atopic dermatitis, asthma, and allergy).

42. Use according to any one of claims 33 to 41 wherein the condition associated with the activity of a serine/threonine protein phosphatase is a neurodegenerative condition.

43. Use according to claim 42 wherein the neurodegenerative condition is a tauopathy.

44. Use according to claim 42 or 43 wherein the neurodegenerative condition is selected from the group consisting of Creutzfeldt-Jakob disease, Huntington's disease, stroke, cerebral ischaemia, dementia associated with stroke or cerebral ischaemia, dementia associated with HIV, disorders associated with excitotoxicity, epilepsy, seizures, schizophrenia, bipolar disorder, depression, mood disorder, multiple sclerosis, acute brain trauma (severe traumatic brain injury) and motor neurone disease.

45. Use according to claim 42 wherein the neurodegenerative condition is selected from the group consisting of presenile dementia, senile dementia, Alzheimer's disease, Parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy (PSP), Pick's disease, primary progressive aphasia, frontotemporal dementia, corticobasal dementia, Parkinson's disease, dementia with Lewy bodies, Down's syndrome, multiple system atrophy, amyotrophic lateral sclerosis (ALS), multiple sclerosis, and Hallervorden-Spatz syndrome.

46. Use according to any one of claims 33 to 35 wherein the condition associated with the activity of a serine/threonine protein phosphatase is a non-tumor disease or disorder associated with aberrant angiogenesis.

47. Use according to claim 46 wherein the non-tumor disease or disorder associated with aberrant angiogenesis is selected from the group consisting of a clinical condition characterised by excessive vascular endothelial cell proliferation, vascular permeability, edema or inflammation comprising macular degeneration, especially age-related macular degeneration comprising wet macular degeneration and dry macular degeneration; a retinopathy such as diabetic retinopathy, ischaemic retinal vein occlusion and retinopathy of prematurity; endometriosis, restenosis, psoriasis and rheumatoid arthritis, brain edema associated with injury, stroke or tumor, edema associated with inflammatory conditions such as psoriasis, and arthritis comprising rheumatoid arthritis, asthma, generalised edema associated with burns, ascites and pleural effusion, macular degeneration, diabetic retinopathy, endometriosis and restenosis.

48. A method of identifying a compound capable of modulating protein serine/threonine phosphatase activity, the method comprising:

(a) contacting the protein serine/threonine phosphatase or an analogue or fragment thereof with a candidate compound under conditions permitting binding of the test compound to the protein serine/threonine phosphatase; and

(b) determining whether the test compound binds to a binding motif on the catalytic subunit of the protein serine/threonine phosphatase or to an analogue or fragment thereof.

49. A method according to claim 48 comprising determining whether the candidate compound modulates protein serine/threonine phosphatase activity.

50. A method according to claim 48 or claim 49, wherein the protein serine/threonine phosphatase is selected from the group consisting of PP1 α, PP1 β, PP1γ1 , PP1γ2, PP2A, PP2B , PP2C, PP4, PP5 and PP6.

51. A method according to claim 50, wherein the protein serine/threonine phosphatase is PP2A.

52. A method according to claim 50, wherein the protein serine/threonine phosphatase is PP5.

53. A method according to any one of claims 48 to 52, wherein the binding motif includes one or more amino acids from any one of the amino acid clusters selected from the group consisting of:

(a) D57, H59, D85, R89, N117, H118, H167, R214, H241;

(b) D57, H59, D85, N117, H167, H241;

(c) 162,163,235,236,250,254,278,282,284; (d) N1 17,H1 18,S120,R121 ,Q122,l123,T124,E188,V189,P190,H191 ,G192,C196, W200,A216,G217;

(e) I14,N18,M66,F69,R7O,G73,K74,S75,Y8O,L99,L1 O3;

(f) F164,L166,G169,L17O,S171 ,I174,I18O,L198,W2O9,F228;

(g) L39,Y86,V97,V101 ,l1 13,E1 19,F146,L149,F150,L153; (h) Y86,T96,V97,L100,V101 ,l113,F150,L153;

(i) Q46,E47,V48,R49,P51 ,V52,N79,Y80,L81 ,K104,R108,E109,R1 10,11 11.T1 12; C) L31 ,A35,L39,L100,V101 ,K104,V105,l1 13,L149,L153;

(k) D57,V58,Q61 ,D64,L65,L68,S261 ,A262,P263,F289;

(I) C55,G56,D57,V58,H59,L81 ,F82,M83,G84,D85,Y86,L100,L1 14,S261 ; (m) D57,H59,D85,R89,N1 17,H1 18,l123,Y127,W200,R214,H241 ,F260,Y265; (n) V28,K29,C32,E33,K36,K144,Y145,D148,L149;

(o) I14,S75,P76,L1 O3,R1O6,Y1 O7,I1 11 ;

(P) L39,T40,D151 ,Y152,L153,R185,L186,Q187;

(q) H191 ,E192,G193,C196,D197,A216,G217,Y218;

(r) Y107,R108,E109,R110; (s) P51 ,V52,T53,D77,T78,N79,Y80,M276,E277,L278,D279 ;

(t) D131 ,L134,R135,Y307,F308;

(u) V244,M245,E246,G247,Y248,N249,W250;

(v) W200,S201 ,D202,S212,P213,R214,A216,G217,Y218,T219,F220;

(w) R89,R214,H241 ,Q242,L243,F260,Y265; (x) F6,W13,S30,L31 ,E33,K34,E37;

(y) L10,D1 1 ,l14,E15,P76,R106,Y107;

(z) G90,Y91 ,Y92,S93,G128,F129,D131 ,E132,R135,K136;

(aa) L17O,S171 ,I174,D175,W2O9,I224,T227,F228;

(dd) P203,D204,D205,R206,G207,G221 ,Q222,D223,Q242,N249,C251 ,H252,D253;

(ee) L183,D184,R185,Q187,P190,P194,H195;

(ff) Y267,P291 ,A292,R294;

(ii) E33,K36,E37;

Cj) V5,F6,T7,K8,E9;

(kk) I21 1 ,S212,G215,A216,G217,Y218;

(II) E226,N229,H230,L234,N255; (mm) T176,L177,D178,R181 ,N232;

(nn) H63,H66,E67,R70,A292,P293,R294;

(oo) H63,Y92,Y267,R294,R295;

(PP) D202, P203, D204, P213.T219, F220,Q242;

(qq) V300,R303,T304,P305; (rr) H63,P293,R294,R295;

(SS) T40,N44,L183,D184,R185,L186; (tt) E42,Q46,V48,K104,R108,E109,l111,T112;

(UU) D204,R206,G210,l211,S212,P213,T219;

(W) D204,R206,l211,P213,T219;

(WW) N18,E19,C20,F62,H62,M63, (XX) R89, N117, H118, 1123, Y127, W200, P213, R214, H241, Q242, L243, F260, Y265;

(yy) N117, S120, R121, Q122, 1123, Y127, E188, V189, P190, H191, W200;

(ZZ) D204, D205, C221, Q222, G251, H252, D253;

(aaa) R89, R214; (bbb) H191.Q122;

(CCC) E188, R121;

(ddd) H252, D253; and

(eee) D290.

54. A method according to wherein the binding motif is a component of a binding complex, the binding complex comprising an accessory atom, ion or molecule.

55. A method according to claim 54 wherein the accessory ion is Mn2+.

56. A method according to any one of claims 48 to 55 comprising isolating the test compound that has been identified as capable of modulating protein serine/threonine phosphatase activity.

57. A method of identifying a compound which is capable of binding to a protein serine/threonine phosphatase, the method comprising:

(a) providing a three dimensional structure of a catalytic subunit of a protein serine/threonine phosphatase, or an analogue or fragment thereof; and

(b) identifying a candidate compound for binding to one or more binding motifs on the catalytic subunit, or the analogue or fragment thereof by performing structure-based drug design with the structure of (a).

58. A method of identifying a candidate compound capable of binding to a protein serine/threonine phosphatase, the method comprising:

(a) identifying a candidate compound that has a conformation and polarity such that it interacts with at least one relevant amino acid residue of one or more binding motifs on a catalytic subunit of the protein serine/threonine phosphatase, or an analogue or fragment thereof.

59. A computer-assisted method for identifying a candidate compound capable of binding to a protein serine/threonine phosphatase, the method comprising:

(a) supplying a computer modelling application with a set of structure coordinates of a molecule or molecular complex, at least a portion of the structural coordinates of the molecule or molecular complex being derived from, or defining the same relative spatial configuration as, at least a portion of the atomic coordinates of one or more binding motifs on a catalytic subunit of the protein serine/threonine phosphatase; .

(b) supplying the computer modelling application with a set of structure coordinates of the candidate compound; and

(c) determining whether the candidate compound is expected to bind to the molecule or molecular complex, wherein binding to the molecule or molecular complex is indicative of potential binding to the catalytic subunit of the protein serine/threonine phosphatase.

60. A computer-assisted method for designing a candidate compound capable of binding to a catalytic subunit of a protein serine/threonine phosphatase, the method comprising:

(a) supplying a computer modelling application with a set of structure coordinates of a molecule or molecular complex, at least a portion of the structural coordinates of the molecule or molecular complex being derived from, or defining the same relative spatial configuration as, at least a portion of the atomic coordinates of one or more of the binding motifs of the catalytic subunit of the protein serine/threonine phosphatase;

(b) supplying the computer modelling application with a set of structure coordinates for the candidate compound;

(c) evaluating the potential binding interactions between the candidate compound and substrate binding pocket of the molecule or molecular complex; (d) structurally modifying the candidate compound to yield a set of structure coordinates for a modified candidate compound; and

(e) determining whether the modified candidate compound is expected to bind to the molecule or molecular complex, wherein binding to the molecule or molecular complex is indicative of potential binding to the catalytic subunit of the protein serine/threonine phosphatase.

61. A computer-assisted method for designing a candidate compound capable of binding to a catalytic subunit of a protein serine/threonine phosphatase de novo, the method comprising:

(b) computationally building a candidate compound represented by a set of structure coordinates; and

62. A method according to claim 57, wherein the three dimensional structure of the catalytic subunit of the protein serine/threonine phosphatase is provided by using the atomic coordinates as publicly disclosed in the RCSB Protein Databank under accession 2iae.

63. A method according to claim 57 or claim 58, wherein the step of identifying a candidate compound for binding to one or more binding motifs on the catalytic subunit is performed using a fitting operation between the three-dimensional structure of the one or more binding motifs and the candidate compound.

64. A method according to any one of claims 57 to 63, wherein the candidate compound demonstrates a docking score of at least about -2.5, and preferably less than about -7.5.

65. A method according to any one of claims 57 to 64, wherein the one or more binding motifs are selected from the group consisting of:

(a) D57, H59, D85, R89, N117, H118, H167, R214, H241;

(b) D57, H59, D85, N117, H167, H241;

(c) 162,163,235,236,250,254,278,282,284; (d) N1 17,H1 18,S120,R121 ,Q122,l123,T124,E188,V189,P190,H191 ,G192, C196,W200,A216,G217; (e) I14,N18,M66,F69,R7O,G73,K74,S75,Y8O,L99,L1O3;

(f) F164,L166,G169,L17O,S171,I174,I18O,L198,W2O9,F228;

C) L31,A35,L39,L100,V101,K104,V105,l113,L149,L153;

(k) D57,V58,Q61 ,D64,L65,L68,S261 ,A262,P263,F289;

(o) I14,S75,P76,L1O3,R1O6,Y1O7,I111;

(P) L39,T40,D151,Y152,L153,R185,L186,Q187;

(q) H191,E192,G193,C196,D197,A216,G217,Y218;

(r) Y107,R108,E109,R110; (s) P51,V52,T53,D77,T78,N79,Y80,M276,E277,L278,D279 ;

(t) D131,L134,R135,Y307,F308;

(u) V244,M245,E246,G247,Y248,N249,W250;

(v) W200,S201,D202,S212,P213,R214,A216,G217,Y218,T219,F220;

(w) R89,R214,H241,Q242,L243,F260,Y265; (X) F6,W13,S30,L31,E33,K34,E37;

(y) L10,D11,l14,E15,P76,R106,Y107;

(Z) G90,Y91,Y92,S93,G128,F129,D131,E132,R135,K136;

(aa) L17O,S171,I174,D175,W2O9,I224,T227,F228;

(dd) P203,D204,D205,R206,G207,G221,Q222,D223,Q242,N249,C251,H252,D253;

(ee) L183,D184,R185,Q187,P190,P194,H195;

(ff) Y267,P291,A292,R294;

(ii) E33,K36,E37;

Cj) V5,F6,T7,K8,E9;

(kk) I211,S212,G215,A216,G217,Y218;

(II) E226,N229,H230,L234,N255; (mm) T176,L177,D178,R181,N232;

(nn) H63,H66,E67,R70,A292,P293,R294; (oo) H63,Y92,Y267,R294,R295;

(PP) D202,P203,D204,P213.T219,F220,Q242;

(qq) V300,R303,T304,P305;

(rr) H63,P293,R294,R295; (SS) T40,N44,L183,D184,R185,L186;

(tt) E42,Q46,V48,K104,R108,E109,l111,T112;

(UU) D204,R206,G210,l21 1 ,S212,P213,T219;

(W) D204,R206,l21 1 ,P213,T219; and

(WW) N18,E19,C20,F62,H62,M63. (XX) R89, N 1 17, H1 18, 1123, Y127, W200, P213, R214, H241 , Q242, L243, F260,

Y265;

(yy) N117, S120, R121, Q122, 1123, Y127, E188, V189, P190, H191, W200;

(ZZ) D204, D205, C221, Q222, G251, H252, D253;

(aaa) R89, R214; (bbb) H191.Q122;

(CCC) E188, R121;

(ddd) H252, D253; and

(eee) D290.

66. A method according to wherein the binding motif is a component of a binding complex, the binding complex comprising an accessory atom, ion or molecule.

67. A method according to claim 66 wherein the accessory ion is Mn2+.

68. A method according to any one of claims 59 to 67 comprising obtaining the candidate compound, contacting the candidate compound with a protein serine/threonine phosphatase, or an analogue, a homolog or a fragment thereof and determining the ability of the candidate compound to modulate protein serine/threonine phosphatase activity.

69. A method according to claim 68, wherein determining the ability of the candidate compound to modulate protein serine/threonine phosphatase activity includes determining the ability of the candidate compound to enhance protein serine/threonine phosphatase activity.

70. A method according to claim 68, wherein determining the ability of the candidate compound to modulate protein serine/threonine phosphatase activity includes determining the ability of the candidate compound to inhibit protein serine/threonine phosphatase activity.

71. A method of screening to identify a compound capable of modulating protein serine/threonine phosphatase activity comprising: contacting a sample containing a PP2AB subunit complex of the protein serine/threonine phosphatase with a compound to be screened; and determining a differential effect of the compound on a PP2AB form of PP2A in the presence and absence of the compound.

72. A method according to claim 71 wherein the compound is capable of selectively regulating the phosphatase activity of the PP2AB form of PP2A.

73. A method according to claim 71 or claim 72 wherein prior to determining the differential effect of the compound on a PP2AB form of PP2A in the presence and absence of the compound, a differential effect of the compound on phosphatase activity of PP2A in general is determined for comparison against phosphatase activity of the PP2AB form.

74. A method according to any one of claims 71 to 73 wherein the differential effect is measured as an increase or decrease in phosphatase activity of the PP2AB compared to a control in the absence of the compound.

75. A method according to any one of claims 71 to 74 wherein the compound to be identified regulates the phosphatase activity by activating the PP2A and/or PP2AB activity.

76. A method according to any one of claims 71 to 75 wherein the differential effect is determined by a measure of dephosphorylation of a phosphopeptide/protein.

77. A method according to claim 76 wherein the phosphopeptide/protein is protein kinase Akt.

78. A method according to any one of claims 71 to 77 wherein the differential effect is measured by dephosphorylation of tau protein.

79. A method according to any one of claims 71 to 78 wherein the PP2AB form of PP2A is selected from the group consisting of PP2ABα, PP2ABβ, PP2ABγ and

PP2AB5.

80. A method according to any one of claims 71 to 79 wherein the sample is a cell sample selected from the group comprising cells, cell lysate, nucleic acids, proteins and peptides.

81. A compound identified by a method according to any one of claims 71 to80.

82. A composition comprising a compound according to claim 77 in combination with a pharmaceutically acceptable carrier.

83. A method of treating a condition associated with protein serine/threonine phosphatase activity in a patient in need, said method comprising administering an effective amount of a composition according to claim 82 to the patient in need.

84. A method of treating a condition associated with protein serine/threonine phosphatase activity in a patient in need, said method comprising: identifying a compound that selectively regulates phosphatase activity of a form of PP2A, said method comprising: contacting a sample containing PP2AB subunit complex with a compound to be screened; and determining a differential effect of the compound on a PP2AB form of PP2A in the presence and absence of the compound; and administering an effective amount of the identified compound to the patient in need.

85. A method according to claim 84 wherein the condition associated with protein serine/threonine phosphatase activity is a hyperproliferative disorder.

86. A method according to claim 85 wherein the hyperproliferative disorder is cancer.

87. A method according to claim 86 wherein the cancer is selected from the group consisting of brain, lung, liver, spleen, kidney, lymph node, small intestine, pancreas, blood cells, colon, stomach, breast, endometrium, prostate, testicle, ovary, skin, head and neck, esophagus, bone marrow and blood cancer; carcinomas (comprising colon, prostate, breast, melanoma, ductal, endometrial, stomach, dysplastic oral mucosa, invasive oral cancer, non-small cell lung carcinoma, transitional and squamous cell, urinary carcinoma); neurological malignancies (comprising neuroblastoma, gliomas).; hematological malignancies, (comprising childhood acute leukaemia, non-Hodgkin's lymphomas, chronic lymphocytic leukaemia, malignant cutaneous T-cells, mycosis fungoides, non-MF cutaneous T-cell lymphoma, lymphomatoid papulosis, T-cell rich cutaneous lymphoid hyperplasia, bullous pemphigoid, discoid lupus erythematosus, lichen planus; sarcomas, melanomas, adenomas; benign lesions such as papillomas, and the like, uterine, testicular and ovarian carcinomas, endometriosis, squamous and glandular epithelial carcinomas of the cervix.

88. A method according to claim 85 wherein the hyperproliferative disorder is a vascular hyperproliferative disorder

89. A method according to claim 88 wherein the vascular hyperproliferative disorder is selected from the group consisting of restenosis , in-stent stenosis, and vascular graft restenosis, neointimal occlusive lesions, atherosclerosis, graft coronary vascular disease after transplantation, vein graft stenosis, peri-anastomatic prosthetic graft stenosis, restenosis after angioplasty or stent placement.

90. A method according to claim 85 wherein the hyperproliferative disorder is selected from the group consisting of a fibrotic disorder, an inflammatory disorder (comprising arthritis), endometriosis; psoriasis, a benign growth disorder (comprising prostate enlargement and lipoma); and an autoimmune disorder (comprising scleroderma, systemic lupus erythematosus, Sjogren's syndrome, atopic dermatitis, asthma, and allergy).

91. A method according to claim 84 or claim 86 wherein the condition associated with protein serine/threonine phosphatase activity is a neurodegenerative condition.

92. A method according to claim 91 wherein the neurodegenerative condition is a tauopathy.

93. A method according to claim 91 or claim 92 wherein the neurodegenerative condition is selected from the group consisting of Creutzfeldt-Jakob disease,

Huntington's disease, stroke, cerebral ischaemia, dementia associated with stroke or cerebral ischaemia, dementia associated with HIV, disorders associated with excitotoxicity, epilepsy, seizures, schizophrenia, bipolar disorder, depression, mood disorder, multiple sclerosis, acute brain trauma (severe traumatic brain injury) and motor neurone disease.

94. A method according to claim 91 wherein the neurodegenerative condition is selected from the group consisting of presenile dementia, senile dementia, Alzheimer's disease, Parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy (PSP), Pick's disease, primary progressive aphasia, frontotemporal dementia, corticobasal dementia, Parkinson's disease, dementia with Lewy bodies, Down's syndrome, multiple system atrophy, amyotrophic lateral sclerosis (ALS), multiple sclerosis, and Hallervorden-Spatz syndrome.

95. A method according to claim 83 or 84 wherein the condition associated with protein serine/threonine phosphatase activity is a non-tumor disease or disorder associated with aberrant angiogenesis.

96. A method according to claim 95 wherein the non-tumor disease or disorder associated with aberrant angiogenesis is selected from the group consisting of a clinical condition characterised by excessive vascular endothelial cell proliferation, vascular permeability, edema or inflammation comprising macular degeneration, especially age-related macular degeneration comprising wet macular degeneration and dry macular degeneration; a retinopathy such as diabetic retinopathy, ischaemic retinal vein occlusion and retinopathy of prematurity; endometriosis, restenosis, psoriasis and rheumatoid arthritis, brain edema associated with injury, stroke or tumor, edema associated with inflammatory conditions such as psoriasis, and arthritis comprising rheumatoid arthritis, asthma, generalised edema associated with burns, ascites and pleural effusion, macular degeneration, diabetic retinopathy, endometriosis and restenosis.