WO2009132397A1

WO2009132397A1 - Methods and agents for modulating the level and/or activity of hif-2 alpha protein

Info

Publication number: WO2009132397A1
Application number: PCT/AU2009/000544
Authority: WO
Inventors: Benjamin James Roberts; Stavros Paltoglou
Original assignee: University Of South Australia
Priority date: 2008-05-01
Filing date: 2009-05-01
Publication date: 2009-11-05

Abstract

The present invention relates to a method of modulating the level and/or activity of a HIF-2 alpha protein. The method includes modulating phosphorylation of the serine residue in the amino acid sequence FQLSPIC in the HIF-2 alpha protein. The invention also relates to agents capable of modulating phosphorylation of the serine residue in the amino acid sequence FQLSPIC in the HIF-2 alpha protein, comprising the amino acid sequence DFQLSPI. These agents are useful in the treatment of unwanted angiogenesis or cancer.

Description

METHODS AND AGENTS FOR MODULATING THE LEVEL AND/OR ACTIVITY OF HIF-2 ALPHA PROTEIN

Priority Claim

This international patent application claims priority to US provisional patent application 61/049,639, the contents of which is hereby incorporated by reference.

Field

The present invention relates to a method of modulating the level and/or activity of a HIF-2 alpha protein.

The present invention also relates to an agent that modulates the level and/or activity of a HIF-2 alpha protein, and to a method of identifying an agent that modulates the level and/or activity of a HIF-2 alpha protein.

Background

Hypoxia Inducible Factor-2 alpha (HIF-2α) is one of a family of proteins that regulate transcription in response to changes in oxygen tension. Other members of this family include Hypoxia Inducible Factor- 1 alpha (HIF- lα) and Hypoxia Inducible Factor -3 alpha (HIF-3α). This family of proteins act as transcriptional activators by forming heterodimers with aryl hydrocarbon receptor nuclear translocator protein, also known as ARNT or HIF- lβ, which bind to their cognate binding elements and activate transcription.

HIF- lβ is a stable protein that is equivalently expressed in normoxia and hypoxia. Unlike HIF-I β, the HIF- lα and HIF-2α proteins are rapidly degraded under normoxic conditions, leading to low levels of transcriptional activity for genes under their control. However, under conditions of reduced oxygen tension the proteins are stabilised, resulting in an induction of transcription of genes under their control. Degradation of the proteins is mediated by an approximately 200-amino acid domain, which has been termed the "oxygen-dependent degradation domain" (ODDD), and appears to involve ubiquitination and subsequent proteasomal digestion.

Recent evidence indicates that the von Hippel Lindau/E3 ligase complex is responsible for the ubiquitination of HIF-I α and HIF-2α, by targeting the proteins for proteosomal degradation via ubiquitination. The interaction between the HIF- lα and HIF-2α proteins and von Hippel Lindau (VHL) appears to involve proline hydroxylation of HIF- lα and HIF-2α, a constitutive reaction during normoxia that maintains the interaction between VHL and the proteins, thus targeting them for degradation. During hypoxia, the catalytic activity of one or more proline hydroxylases may be diminished through a reduction in available oxygen, thus disassociating VHL from the proteins, resulting in their stabilisation and translocation to the nuclear compartment.

One of the primary roles of the family of Hypoxia Inducible Factors is the regulation of vascular development via changes involving reduced oxygen tension. Trans-activation of genes involved in vascular development containing cognate binding elements for HIF- lα and HIF-2α at low oxygen tension results in increased vascularisation and oxygenation of tissues starved of oxygen.

Because of their role in regulating vascularisation, there has been significant effort applied to studying how changes in the expression and/or activity of the Hypoxia Inducible Factors may be used to control angiogenesis, the process by which new blood vessels are formed.

In this regard, angiogenesis is not only an essential part of many normal biological processes such as embryonic development, the formation of endometrium and in wound healing, but persistent and/or dysregulated blood vessel growth is also associated with numerous pathological conditions. For example, the growth of tumours beyond a few millimetres in size can only occur upon neovascularisation of the tumour. New blood vessel growth also promotes the metastasis of tumours. In diabetic retinopathy, the invasion of the retina by new blood vessels leads to blindness in patients. Neovascularization of the ocular apparatus is a major cause of blindness and such neovascularization is responsible for a number of diseases of the eye. Neovascularisation is also involved in the pathogenesis of rheumatoid arthritis.

In diseases, conditions and states associated with excessive, undesired or dysregulated blood vessel growth, the inhibition of angiogenesis provides a possible therapeutic avenue for treatment. On the other hand, in the case of conditions such as ischemia or wound healing, the promotion of angiogenesis provides a possible therapeutic avenue for treatment.

HIF-2α shares approximately 48% homology with HIF- lα, is induced by hypoxia, and up-regulates a similar though not identical set of hypoxia-responsive genes. However, there are some differences in the expression and regulation of HIF-2α as compared to HIF- lα. Most notably, HIF- lα and HIF-2α are frequently expressed to different levels in different tissues, and using inhibitory RNA approaches, have been shown to regulate different genes in vivo, suggesting they are factors with discrete biological roles. By way of confirmation, HIF-2α gene deletion offers different phenotypes to that seen with HIF- lα.

Although most studies have concentrated on the role of HIF- lα in regulating angiogenesis, recent studies have highlighted that HIF-2α may in fact play a more significant role in controlling angiogenesis than initially anticipated. For example, HIF- 2α overexpresssion or substitution into the HIF- lα locus, rather than HIF- lα overexpression, has been shown to result in renal cell cancer growth. Paradoxically, HIF- lα overexpression appears to have a tumour inhibiting effect. Additionally, HIF- 2α has been shown to stain heavily in tumour associated macrophages, suggesting it may also respond to, or be associated with, inflammation in addition to low oxygen.

The ability to regulate the level and/or activity of HIF-2α provides an avenue for therapeutic intervention in diseases, conditions and states associated with, or responding to, altered levels and/or activity of HIF-2α. A reference herein to a patent document or other matter which is given as prior art is not to be taken as an admission that that document or matter was known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims.

Summary

The present invention provides a method of modulating the level and/or activity of a HIF-2 alpha protein, the method including modulating phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in the HIF-2 alpha protein.

The present invention also provides a method of modulating angiogenesis in a biological system, the method including modulating phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in a HIF-2 alpha protein in one or more cells in the biological system.

The present invention also provides a method of preventing and/or treating a disease, condition or state associated with undesired and/or uncontrolled angiogenesis in a subject, the method including administering to the subject an agent that modulates phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in a HIF-2 alpha protein.

The present invention also provides use of an agent that modulates phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in a HIF-2 alpha protein in the preparation of a medicament for modulating angiogenesis.

The present invention also provides an agent that modulates the level and/or activity of a HIF-2 alpha protein by modulating phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) sin a HIF-2 alpha protein.

The present invention also provides an agent that modulates phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in a HIF-2 alpha protein, the agent including an amino acid sequence DFQLSPI (SEQ ID NO.23), or a functional variant thereof.

The present invention also provides an agent that modulates angiogenesis, the agent modulating phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.1) in a HIF-2 alpha protein.

The present invention also provides an agent that modulates angiogenesis, the agent including an amino acid sequence DFQLSPI (SEQ ID NO.23), or a functional variant thereof.

The present invention also provides an isolated polypeptide consisting of an amino acid sequence selected from the group of amino acid sequences as provided by SEQ ID Nos 3 to 25, or a functional variant thereof.

The present invention also provides a method of identifying an agent that modulates the level and/or activity of HIF-2 alpha protein, the method including identifying an agent that modulates phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in a HIF-2 alpha protein.

The present invention also provides a method of identifying an agent that modulates angiogenesis, the method including identifying an agent that modulates phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in a HIF-2 alpha protein.

The present invention also provides a method of identifying an agent that modulates the level and/or activity of a HIF-2 alpha protein, the method including: providing a candidate compound including an amino acid sequence DFQLSPI

(SEQ ID NO.23), or a functional variant thereof; determining the ability of the candidate compound to modulate the level and/or activity of a HIF-2 alpha protein in a cell; and identifying the candidate compound as an agent that modulates the level and/or activity of a HIF-2 alpha protein in a cell. The present invention also provides a method of identifying an agent that modulates angiogenesis, the method including: providing a candidate compound including an amino acid sequence DFQLSPI

(SEQ ID NO.23), or a functional variant thereof; determining the ability of the candidate compound to modulate angiogenesis; and identifying the candidate compound as an agent that modulates angiogenesis.

The present invention also provides a method of identifying an agent that modulates phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in a HIF-2 alpha protein, the method including: providing a candidate compound including an amino acid sequence DFQLSPI

(SEQ ID NO.23), or a functional variant thereof; and determining the ability of the candidate compound to modulate phosphorylation of the serine residue; and identifying the candidate compound as an agent that modulates phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in a

HIF-2 alpha protein.

The present invention arises from investigations into the variable inducibility of HIF-I α and HIF-2α across numerous cell lines.

In the present studies, it has been recognised that in some cell lines there is little or no discernible increase in HIF-2α following hypoxia or DPP treatment for up to 6 hours, while under similar conditions a significant increase in HIF- lα expression is observed. In addition, it is possible to readily detect HIF-2α during normoxia, suggesting stabilization of the HIF-2α molecule.

It has been demonstrated in the present studies that phosphorylation of the HIF-2α protein at the conserved serine 543 residue under normoxia results in stabilization of the protein, while phosphorylation targets the protein for degradation under hypoxia. Phosphorylation of this serine residue inhibits ubiquitination of the protein by the VHL/E3 ligase complex. Thus, the degradation of HIF-2α may be regulated by phosphorylation of a specific serine residue on the protein.

It is also contemplated that this novel post-translation modification that stabilizes HIF- 2α under normoxic conditions may also occur constitutively in transformed and tumour cells. Thus, regulation of this modification may be used to control angiogenesis and other biological effects under the control of HIF-2α.

Various terms that will be used throughout the specification have meanings that will be well understood by a skilled addressee. However, for ease of reference, some of these terms will now be defined.

The term "HIF-2 alpha protein" as used throughout the specification is to be understood to mean a member of the HIF-2 alpha family of proteins, and which has a serine residue in the position corresponding to amino acid 543 in the human protein. This serine residue is located in the conserved amino acid sequence FQLS^PI (SEQ ID NO.l).

Generally, a HIF-2 alpha protein will include (i) a basic helix-loop-helix dimerization domain; (ii) a PER-ARNT-SIM domain; (iii) an oxygen-dependent degradation domain; (iv) a transactivation domain; and (iv) a nuclear localization signal. It will be appreciated that the term includes within its scope a natural protein, a variant of a natural protein (including a deletion or fusion of the protein), or a synthetic protein.

In most cases, a HIF-2 alpha protein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% amino acid sequence identity to human HIF-2 alpha protein (UniProtKB/Swiss-Prot entry Q99814).

When comparing amino acid sequences to calculate a percentage identity, the compared sequences should be compared over a comparison window of at least 200 amino acid residues, at least 400 amino acid residues, at least 600 amino acid residues, at least 800 amino acid residues or over the full length of UniProtKB/Swiss-Prot entry Q99814. The comparison window may comprise additions or deletions (ie. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms such as the BLAST family of programs as, for example, disclosed by Altschul et al. (Nucl. Acids Res. 25: 3389-3402, 1997). A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al. ("Current Protocols in Molecular Biology" John Wiley & Sons Inc, 1994-1998, Chapter 15, 1998).

Examples of HIF-2 alpha proteins from various other species and their accession numbers include mouse (AAB41496), rat (CAB96612), pig (ABN48508), null (NP 777150), rhesus monkey (XP OOl 113007), chimpanzee (XP OO 1147074), sheep (AAR37391), chicken (AAD38358), and quail (AAF21052).

HIF-2 alpha proteins from other species may be readily identified, for example by use of the BLAST algorithm, which determines the extent of homology between two nucleotide sequences (blastn) or the extent of homology between two amino acid sequences (blastp). BLAST identifies local alignments between the sequences in the database and predicts the probability of the local alignment occurring by chance. The BLAST algorithm is as described in Altschul et al. (1990, supra).

The term "biological system" as used throughout the specification is to be understood to mean any multi-cellular system, and includes for example isolated groups of cells, tissue or organs, and whole organisms (including a human or animal subject).

The term "variant" as used throughout the specification is to be understood to mean an amino acid sequence of a polypeptide or protein that is altered by one or more amino acids. The variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties to the replaced amino acid (e.g., replacement of leucine with isoleucine). A variant may also have "non-conservative" changes (e.g., replacement of a glycine with a tryptophan) or a deletion and/or insertion of one or more amino acids. A variant may also be a form of the protein that has one or more deleted amino acids (eg a truncated form of the protein), and/or a form of the protein that has one or more additional exogenous amino acids (eg a form of the protein fused to another polypeptide sequence). It will be appreciated that a variant will therefore include within its scope a fragment of a protein.

Generally, the variant will be a functional variant, that is, a variant that retains the functional ability of the progenitor protein.

The term "nucleic acid" as used throughout the specification is to be understood to mean to any oligonucleotide or polynucleotide. The nucleic acid may be DNA or RNA and may be single stranded or double stranded. The nucleic acid may be any type of nucleic acid, including a nucleic acid of genomic origin, cDNA origin (ie derived from a mRNA), derived from a virus, or of synthetic origin.

In this regard, an oligonucleotide or polynucleotide may be modified at the base moiety, sugar moiety, or phosphate backbone, and may include other appending groups to facilitate the function of the nucleic acid. The oligonucleotide or polynucleotide may be modified at any position on its structure with constituents generally known in the art. For example, an oligonucleotide may include at least one modified base moiety which is selected from the group including 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5- iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyliydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1- methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2- methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta D- mannosylqueosine, S'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6- isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil5- oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3- (3- amino-3-N-2-carboxypropyl) uracil, (acp3) w, and 2,6-diaminopurine.

The oligonucleotide or polynucleotide may also include at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2- fluoroarabinose, xylulose, and hexose. In addition, the oligonucleotide or polynucleotide may include at least one modified phosphate backbone, such as a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or any analogue thereof.

The term "subject" as used throughout the specification is to be understood to mean a multicellular organism for which the degradation of HIF-2α is regulated by phosphorylation of a specific serine residue on the protein, corresponding to the serine reside at amino acid residue 543 of the human protein. In this regard, it is envisaged that the invention will be applicable in species ranging from birds to humans. For example, the subject may be a human or other mammal, a primate, a livestock animal (eg. a horse, a cow, a sheep, a pig, or a goat), a companion animal (eg. a dog, cat), a laboratory test animal (eg. a mouse, a rat, a guinea pig, a bird, a rabbit), an animal of veterinary significance, or an animal of economic significance.

The term "modulate" as used throughout the specification is to be understood to mean a change or alteration in the progress of a process, including a change or alteration in any one or more of the start, continuation or termination of a process.

The term "inhibit" as used throughout the specification is to be understood to mean a reduction in the progress of a process, including any one or more of the start, continuation or termination of a process.

The term "promote" as used throughout the specification is to be understood to mean an increase in the progress of a process, including any one or more of the start, continuation or termination of a process.

The term "angiogenesis" as used throughout the specification is to be understood to mean the generation of new blood vessels ("neovascularization"), for example into a tissue or organ. The term "anti-angiogenic agent" as used throughout the specification is to be understood to mean an agent that has the capacity to inhibit angiogenesis in a biological system.

The term "pro-angiogenic agent" as used throughout the specification is to be understood to mean an agent that has the capacity to promote angiogenesis in a biological system.

General Description

As described above, in one embodiment the present invention provides a method of modulating the level and/or activity of a HIF-2 alpha protein, the method including modulating phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in the HIF-2 alpha protein.

In this regard, it will be understood that the level and/or activity of the HIF-2 alpha protein may be modulated in one or more cells, or alternatively, be modulated in a cell- free system.

In one embodiment, the level and/or activity of the HIF-2 alpha protein is modulated in one or more cells.

In one specific embodiment, increasing phosphorylation of the serine residue increases the level and/or activity of the HIF-2 alpha protein in a cell under normoxic conditions.

In an alternative embodiment, decreasing phosphorylation of the serine decreases the level and/or activity of the HIF-2 alpha protein in a cell under normoxic conditions.

In another specific embodiment, increasing phosphorylation of the serine residue decreases the level and/or activity of the HIF-2 alpha protein in a cell under hypoxic conditions. In another alternative embodiment, decreasing phosphorylation of the serine residue increases the level and/or activity of the HIF-2 alpha protein in a cell under hypoxic conditions.

It will be appreciated that in the case of the modulation of the level and/or activity of a HIF-2 alpha protein in a cell, the HIF-2 alpha protein may be an endogenous and/or an exogenous HIF-2 alpha protein present in the cell.

In one embodiment, the HIF-2 alpha protein is an endogenous HIF-2 alpha protein present in the cell.

Methods for determining the level and/or activity of HIF-2 alpha are known in the art. For example, determination of the level and/or activity of a HIF-2 alpha protein in a cell may include the use of endogenous genes or reporter genes regulated by a cognate binding element for HIF-2 alpha proteins.

Methods for determining the extent of phosphorylation of the HIF-2 alpha include, for example, detection of the protein by use of a specific HIF-2 alpha antibody in conjunction with the determination of the extent of phosphorylation using a phosphoserine antibody. Antibodies that specifically detect HIF-2 alpha protein, and antibodies that specifically detect phosphoserine residues, are known in the art.

As described previously herein, the method may be used to modulate the level and/or activity of a HIF-2 alpha protein in one or more cells. For example, the one or more cells may be present in a biological system, such as an entire human or animal subject. Alternatively, the one or more cells may be one or more cells present in vitro, such as a cell in tissue culture.

In one embodiment, the cell is a cell in a biological system.

In this regard, the term "biological system" is to be understood to mean any multicellular system, including isolated groups of cells to whole organisms. For example, the biological system may be cells in a tissue or organ, or cells in an entire human or animal subject, including a human or animal susceptible to, suffering from, or in need of treatment for, a disease, condition or state that would benefit from modulation of the level and/or activity of a HIF-2 alpha protein.

In one embodiment, the biological system is a human or animal subject.

In this regard, the term "subject" is generally to be understood to mean a multicellular organism for which the degradation of HIF-2α is regulated by phosphorylation of a specific serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) of the protein. It is envisaged that the invention will be applicable in species ranging from birds to humans. For example, the subject may be a human or other mammal, a primate, a livestock animal (eg. a horse, a cow, a sheep, a pig, or a goat), a companion animal (eg. a dog, cat), a laboratory test animal (eg. a mouse, a rat, a guinea pig, a bird, a rabbit), an animal of veterinary significance, or an animal of economic significance.

For example, in the case of a human or animal subject, the subject may be susceptible to, suffering from, or in need of treatment for: (i) a disease, condition or state associated with an altered level and/or activity of a HIF-2 alpha protein; (ii) a disease, condition or state associated with dysregulation of HIF-2 alpha protein degradation; (iii) a disease, condition or state that would benefit from modulation of the level and/or activity of a HIF-2 alpha protein; or (iv) a disease, condition or state that would benefit from modulation of angiogenesis.

In one embodiment, the human or animal subject is a subject susceptible to, or suffering from, a disease, condition or state that would benefit from inhibiting angiogenesis. For example, the subject may be susceptible to, or suffering from, undesired or uncontrolled angiogenesis.

In this regard, diseases, condition or states associated with undesired or uncontrolled angiogenesis include growth of solid tumours; angiofibroma; corneal neovascularisation; retinal/choroidal neovascularization; arteriovenous malformations; arthritis, including rheumatoid arthritis, lupus and other connective tissue disorders; Osier-Weber syndrome; atherosclerotic plaques; psoriasis; pyogenic granuloma; retrolental fibroplasias; scleroderma; granulations, hemangioma; trachoma; hemophilic joints; vascular adhesions and hypertrophic scars; diseases associated with chronic inflammation including sarcoidosis and inflammatory bowel diseases such as Crohn's disease and ulcerative colitis.

Examples of cancers include carcinoma, bladder cancer, bone cancer, brain tumours, breast cancer, cervical cancer, colorectal cancer including cancer of the colon, rectum, anus, and appendix, cancer of the oesophagus, Hodgkin's disease, kidney cancer, cancer of the larynx, leukaemia, liver cancer, lung cancer, lymphoma, melanoma, moles and dysplastic nevi, multiple myeloma, muscular cancer, non-Hodgkin's lymphoma, oral cancer, ovarian cancer, cancer of the pancreas, prostate cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, teratoma, thyroid cancer, cancer of the uterus, adenocarcinoma, and neuroblastoma.

In addition, angiogenesis is also involved in reproduction and wound healing. In reproduction, angiogenesis is an important step in ovulation and also in implantation of the blastula after fertilization. Accordingly, the prevention and/or inhibition of angiogenesis may be used to induce amenorrhea, to block ovulation, or to prevent implantation by the blastula.

In wound healing, excessive repair or fibroplasia can be a detrimental side effect of surgical procedures and may be caused or exacerbated by angiogenesis. Adhesions are also a frequent complication of surgery and lead to problems such as small bowel obstruction. Inhibition of angiogenesis may be used to reduce these problems.

In another embodiment, the human or animal subject is a subject susceptible to, or suffering from, a disease, condition or state that would benefit from promoting angiogenesis.

For example, under conditions of tissue ischemia (eg cardiac ishemia) it may be beneficial to increase neovascularisation of the tissue or organ in a subject. Under some circumstance it may also be beneficial to promote angiogenesis during wound healing. Modulation of the phosphorylation of a HIF-2 alpha protein may also be used to modulate degradation of the protein.

In one embodiment, modulation of phosphorylation of the serine residue may be used to modulate degradation of the HIF-2 alpha protein in a cell.

For example, under normoxic conditions, increasing phosphorylation of the serine residue may be used to decrease degradation of the HIF-2 alpha protein, while decreasing phosphorylation of the serine may be used to promote degradation of the HIF-2 alpha protein.

Under hypoxic conditions, increasing phosphorylation of the serine residue may be used to promote the degradation of the HIF-2 alpha protein, while decreasing phosphorylation of the serine residue may be used to decrease the degradation of the HIF-2 alpha protein.

Modulation of the phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in the HIF-2 alpha protein may be achieved by a suitable method.

For example, modulation of phosphorylation of the serine residue may include modulating the activity of a mitogen-activated kinase in the cell that phosphorylates the appropriate serine residue, such as modulating the activity of p38 MAP kinase.

In one specific embodiment, the modulation of phosphorylation of the serine residue in the HIF-2 alpha protein in a cell includes exposing the cell to an agent that modulates phosphorylation of the serine residue in the HIF-2 alpha protein in the cell.

Examples of agents in the various embodiments of the present invention include a drug, a small molecule, a nucleic acid, an oligonucleotide, a peptide, a polypeptide, a protein, an enzyme, a polysaccharide, a glycoprotein, a lipid, an antibody or a part thereof, and an aptamer. In one embodiment, the agent decreases phosphorylation of the serine residue directly or indirectly, such as an agent that is an inhibitor of a kinase that is involved in phosphorylation of the residue, or an agent that acts as a competitive inhibitor of phosphorylation of the serine reside.

In one embodiment, the agent includes the amino acid sequence DFQLSPI (SEQ ID NO.23), or a functional variant thereof.

In this regard, a functional variant of the amino acid sequence DFQLSPI (SEQ ID NO.23) may be identified by determination of the ability of an agent including a variant of this amino acid sequence to modulate phosphorylation of a HIF-2 alpha protein at the specific serine residue. An example of a suitable assay is as described in Example 6.

Possible functional variants include: (i) a variant that has one or more "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties to the replaced amino acid; and/or (ii) a variant that has one or more "non-conservative" changes; and/or (iii) a variant that has a deletion and/or insertion of one or more amino acids.

Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine. Under some circumstances, substitutions within the aliphatic group alanine, valine, leucine and isoleucine are also considered as conservative. Sometimes substitution of glycine for one of these can also be considered conservative. Other conservative interchanges include those within the aliphatic group aspartate and glutamate; within the amide group asparagine and glutamine; within the hydroxyl group serine and threonine; within the aromatic group phenylalanine, tyrosine and tryptophan; within the basic group lysine, arginine and histidine; and within the sulfur-containing group methionine and cysteine. Sometimes substitution within the group methionine and leucine can also be considered conservative. The present invention contemplates functional variants of the amino acid sequence DFQLSPI (SEQ ID NO.23) with one or more of the above described amino acid substitutions. Example of agents including the amino acid sequence DFQLSPI (SEQ ID NO.23) are agents including one or more of the following amino acid sequences:

MDGEDFQLSPICPEERLLAE (SEQ ID NO. 3) MDGEDFQLSPICPEERLLA (SEQ ID NO. 4) MDGEDFQLSPICPEERLL (SEQ ID NO. 5) MDGEDFQLSPICPEERL (SEQ ID NO. 6) MDGEDFQLSPICPEER (SEQ ID NO. 7) MDGEDFQLSPICPEE (SEQ ID NO. 8) MDGEDFQLSPICPE (SEQ ID NO. 9) MDGEDFQLSPICP (SEQ ID NO. 10) MDGEDFQLSPIC (SEQ ID NO. 11) EDFQLSPICPEER (SEQ ID NO. 12) EDFQLSPICPEE (SEQ ID NO. 13) EDFQLSPICPE (SEQ ID NO. 14) DFQLSPICPE (SEQ ID NO. 15) EDFQLSPICPEERLLAEN (SEQ ID NO. 16) YIPMDGEDFQLSPIC (SEQ ID NO. 17) IPMDGEDFQLSPICPEER (SEQ ID NO. 18) PMDGEDFQLSPICPEER (SEQ ID NO. 19) YIPMDGEDFQLSPICPEER (SEQ ID NO. 20) IPMDGEDFQLSPICPEER (SEQ ID NO. 21) PMDGEDFQLSPICPEER (SEQ ID NO. 22) DFQLSPI (SEQ ID NO. 23) DFQLSPIC (SEQ ID NO. 24) DFQLSPICP (SEQ ID NO.25)

In one embodiment, the agent is a polypeptide including one or more of amino acid sequences selected from the group of amino acid sequences SEQ ID NOs. 3 to 25, or a functional variant thereof. In one specific embodiment, the agent is a polypeptide consisting of one of the amino acid sequences SEQ ID NOs. 3 to 25, or a functional variant thereof. Methods for producing polypeptides are known in the art.

In one embodiment, the polypeptide is myristoylated. Methods for producing myristoylated polypeptides are known in the art.

In the case of an agent that is an inhibitor of a kinase that is involved in phosphorylation of the serine residue, in one embodiment the agent is an inhibitor of a p38 Map kinase.

Examples of agents that are inhibitors of p38 Map kinases include SB 203580, SB 239063, and SK&F 86002. Inhibitors of p38 Map kinase inhibitors are generally as described in the Handbook of Experimental Pharmacology (2005) - "Inhibitors of Protein Kinases and Protein Phosphatases" ed. Lorenzo A. Pinna and Patricia T.W. Cohen, Springer Berlin Heidelberg.

In an alternative embodiment, the agent increases phosphorylation of the serine residue. For example, the agent may promote the activity of a kinase that is involved in phosphorylation of the residue.

The present invention also provides an agent that modulates the level and/or activity of a HIF-2 alpha protein by modulating phosphorylation of the protein.

Accordingly, in another embodiment the present invention provides an agent that modulates the level and/or activity of a HIF-2 alpha protein by modulating phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in a HIF-2 alpha protein.

The present invention also provides a pharmaceutical composition including such an agent, and the use of such an agent in the preparation of a medicament.

In one embodiment, the agent includes an amino acid sequence DFQLSPI (SEQ ID NO.23), or a functional variant thereof. In one specific embodiment, the agent is a polypeptide including one or more of amino acid sequences selected from the group of amino acid sequences SEQ ID NOs. 3 to 25, or a functional variant thereof.

In another specific embodiment, the agent is a polypeptide consisting of one of the amino acid sequences SEQ ID NOs. 3 to 25, or a functional variant thereof. Methods for producing polypeptides are known in the art.

The present invention also provides an agent that modulates phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in a HIF-2 alpha protein.

Accordingly, in another embodiment the present invention provides an agent that modulates phosphorylation of the serine residue in the amino acid sequence FQLSPIC in a HIF-2 alpha protein, the agent including an amino acid sequence DFQLSPI (SEQ ID NO.23), or a functional variant thereof.

Examples of agents are as previously described herein.

In one specific embodiment, the agent is a polypeptide including one or more of amino acid sequences selected from the group of amino acid sequences SEQ ID NOs. 3 to 25, or a functional variant thereof.

In another specific embodiment, the agent is a polypeptide consisting of one of the amino acid sequences SEQ ID NOs. 3 to 25, or a functional variant thereof. Methods for producing polypeptides are known in the art. Modulation of phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in the HIF-2 alpha protein may also be used to modulate angiogenesis in a biological system.

Accordingly, in another embodiment the present invention provides a method of modulating angiogenesis in a biological system, the method including modulating phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in the HIF-2 alpha protein in one or more cells in the biological system.

In one embodiment, the modulation of the phosphorylation of the serine residue includes exposing one or more cells in the biological system to an agent that modulates phosphorylation of the serine residue in the HIF-2 alpha protein.

In one embodiment, the angiogenesis in the biological system is inhibited.

In one embodiment, the agent includes an amino acid sequence DFQLSPI (SEQ ID NO.23) or a functional variant thereof. Examples of such agents are as previously described herein.

As described previously herein, uncontrolled or undesired angiogenesis is associated with a number of diseases, conditions or states, and thus modulating phosphorylation of the HIF-2 alpha protein may be used as a therapeutic route to control such angiogenesis.

As discussed previously herein, diseases, condition or states associated with undesired or uncontrolled angiogenesis include for example growth of solid tumours; angiofibroma; corneal neovascularisation; retinal/choroidal neovascularization; arteriovenous malformations; arthritis, including rheumatoid arthritis, lupus and other connective tissue disorders; Osier- Weber syndrome; atherosclerotic plaques; psoriasis; pyogenic granuloma; retrolental fibroplasias; scleroderma; granulations, hemangioma; trachoma; hemophilic joints; vascular adhesions and hypertrophic scars; diseases associated with chronic inflammation including sarcoidosis and inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Examples of cancers are as previously described herein.

The prevention and/or inhibition of angiogenesis may also be used to induce amenorrhea, to block ovulation, or to prevent implantation by the blastula.

In wound healing, excessive repair or fibroplasia can be a detrimental side effect of surgical procedures and may be caused or exacerbated by angiogenesis. Adhesions are also a frequent complication of surgery and lead to problems such as small bowel obstruction. Thus, inhibition of angiogenesis may be used to reduce these problems.

In an alternative embodiment, the angiogenesis in the biological system is promoted.

For example, under conditions of tissue ischemia (eg cardiac ishemia) it may be beneficial to increase neovascularisation of the tissue or organ in a subject. Another circumstance where promoting angiogenesis may be beneficial is wound healing.

In one embodiment, the modulation of angiogenesis occurs in a human or animal subject.

In one specific embodiment, the modulation of phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in a HIF-2 alpha protein may be used to prevent and/or treat a disease, condition or state associated with undesired and/or uncontrolled angiogenesis in a subject.

Accordingly, in another embodiment the present invention provides a method of preventing and/or treating a disease, condition or state associated with undesired and/or uncontrolled angiogenesis in a subject, the method including administering to the subject an agent that modulates phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in a HIF-2 alpha protein.

Examples of diseases, conditions and states associated with undesired and/or uncontrolled angiogenesis are as previously described herein. The agent that modulates phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in a HIF-2 alpha protein may also be used in the preparation of a medicament for modulating angiogenesis.

Accordingly, in another embodiment the present invention provides use of an agent that modulates phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in a HIF-2 alpha protein in the preparation of a medicament for modulating angiogenesis.

Examples of agents are as previously described herein. For example, the agent may include an amino acid sequence DFQLSPI (SEQ ID NO.23), or a functional variant thereof.

The present invention also provides an agent that modulates angiogenesis by modulating phosphorylation of the HIF-2 alpha protein.

Accordingly, in another embodiment the present invention provides an agent that modulates angiogenesis, the agent modulating phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in a HIF-2 alpha protein.

The present invention also provides a pharmaceutical composition including such an agent.

In one embodiment, the agent is an anti-angiogenic agent.

In an alternative embodiment, the agent is a pro-angiogenic agent.

In one embodiment, the agent includes an amino acid sequence DFQLSPI (SEQ ID NO.23), or a functional variant thereof.

In one specific embodiment, the agent is a polypeptide including one or more of amino acid sequences selected from the group of amino acid sequences SEQ ID NOs. 3 to 25, or a functional variant thereof. In another specific embodiment, the agent is a polypeptide consisting of one of the amino acid sequences SEQ ID NOs. 3 to 25, or a functional variant thereof. Methods for producing polypeptides are known in the art.

The present invention also provides an agent including an amino acid sequence of DFQLSPI (SEQ ID NO.23), or a functional variant thereof, that has the ability to modulate angiogenesis.

Accordingly, in another embodiment the present invention provides an agent that modulates angiogenesis, the agent including an amino acid sequence DFQLSPI (SEQ ID NO.23), or a functional variant thereof.

In one embodiment, the agent is an anti-angiogenic agent.

In an alternative embodiment, the agent is a pro-angiogenic agent.

The agent including an amino acid sequence DFQLSPI (SEQ ID NO.23), or a functional variant thereof, may also be used in the preparation of a medicament for modulating angiogenesis. Accordingly, in another embodiment the present invention provides use of an agent including an amino acid sequence DFQLSPI (SEQ ID NO.23), or a functional variant thereof, in the preparation of a medicament for modulating angiogenesis.

The present invention also provides an isolated polypeptide with an amino acid sequence selected from the group of amino acids sequences SEQ ID Nos 3 to 25.

Accordingly, in another embodiment the present invention provides an isolated polypeptide consisting of an amino acid sequence selected from the group of amino acid sequences as provided by SEQ ID Nos 3 to 25, or a functional variant thereof.

In this regard, the term "isolated" as used throughout the specification is to be understood to mean an entity, for example a polypeptide, nucleic acid, antibody or a cell, which is removed from its natural environment.

The polypeptides may also be administered to a subject to prevent and/or treat a disease, condition or state associated with undesired and/or uncontrolled angiogenesis.

The polypeptides may also be used in the preparation of a medicament, such as a medicament for inhibiting angiogenesis, or a medicament for preventing and/or treating a disease, condition or state associated with undesired and/or uncontrolled angiogenesis.

Methods for exposing an agent to a cell in vitro or in vivo are known in the art.

In the case of an agent delivered to one or more cells in a biological system, the agent can be administered by a method known in the art, including direct delivery of the agent to the desired site of action, and/or administration of the agent to the biological system, including the use of viral delivery and gene therapy techniques.

In the case of an agent delivered to a biological system in the various embodiments of the present invention, the amount of the agent to be used is not particularly limited, so long as it is an effective amount and in such a form that generally exhibits a useful or therapeutic effect. The term "therapeutically effective amount" is the quantity which, when administered to a subject in need of treatment, improves the prognosis and/or state of the subject. The amount to be administered to a subject will depend on the particular characteristics of the disease, condition or state to be treated, the mode of administration, and the characteristics of the subject, such as general health, other diseases, age, sex, genotype, and body weight. A person skilled in the art will be able to determine appropriate dosages depending on these and other factors.

The agent may be delivered in a form and at a concentration suitable to allow the agent to reach the desired site of action and have the desired effect.

The administration of the agent to a subject may be within any time suitable to produce the desired effect. In a human or animal subject, the agent may be administered orally, parenterally, topically or by any other suitable means, and therefore transit time of the agent must be taken into account.

For example, in the case of inhibiting angiogenesis, the administration of the agent to a subject may be at one or more of prior to the start of angiogenesis, and/or concurrently with angiogenesis occurring. For example, in the case of inhibiting angiogenesis associated with a solid cancer, the administration of the agent may be before and/or during the growth of a tumour (primary and/or secondary tumours), and/or before or after resection of a tumour (primary and/or secondary tumours).

The agent in the various embodiments of the present invention may be administered to the subject in a suitable form.

As discussed previously herein, for example administration and delivery of the agent may be by the intravenous, intraperitoneal, subcutaneous, intramuscular, oral, topical route, or by direct injection into the desired site of action. The mode and route of administration in most cases will depend on the type of disease, condition or state being treated. The dosage form, frequency and amount of dose will depend on the mode and route of administration. Typically an injectable composition will be administered in an amount of between 5 mg/m² and 500 mg/m², for example between 10 mg/m² and 200 mg/m² Typically an orally administered composition will be administered in an amount of between 5 mg and 5 g, for example between 50 mg and 1 g.

For example, an effective amount of the agent typically ranges between about 0.1 mg/kg body weight per day and about 1000 mg/kg body weight per day, usually between 1 mg/kg body weight per day and 100 mg/kg body weight per day.

The administration of a composition including such agents may also include the use of one or more pharmaceutically acceptable additives, including pharmaceutically acceptable salts, amino acids, polypeptides, polymers, solvents, buffers, excipients, preservatives and bulking agents, taking into consideration the particular physical, microbiological and chemical characteristics of the agent to be administered.

For example, the agent can be prepared into a variety of pharmaceutically acceptable compositions in the form of, e.g., an aqueous solution, an oily preparation, a fatty emulsion, an emulsion, a lyophilised powder for reconstitution, etc. and can be administered as a sterile and pyrogen free intramuscular or subcutaneous injection or as injection to an organ, or as an embedded preparation or as a transmucosal preparation through nasal cavity, rectum, uterus, vagina, lung, etc. The composition may be administered in the form of oral preparations (for example solid preparations such as tablets, caplets, capsules, granules or powders; liquid preparations such as syrup, emulsions, dispersions or suspensions).

Compositions containing the agent may also contain one or more pharmaceutically acceptable preservative, buffering agent, diluent, stabiliser, chelating agent, viscosity- enhancing agent, dispersing agent, pH controller, solubility modifying agent or isotonic agent. These excipients are well known to those skilled in the art.

Examples of suitable preservatives are benzoic acid esters of para-hydroxybenzoic acid, propylene glycol, phenols, phenylethyl alchohol or benzyl alcohol. Examples of suitable buffers are sodium phosphate salts, citric acid, tartaric acid and the like. Examples of suitable stabilisers are, antioxidants such as alpha-tocopherol acetate, alpha- thioglycerin, sodium metabisulphite, ascorbic acid, acetylcysteine, 8-hydroxyquinoline, and chelating agents such as disodium edetate. Examples of suitable viscosity enhancing agents, suspending or dispersing agents are substituted cellulose ethers, substituted cellulose esters, polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycols, carbomer, polyoxypropylene glycols, sorbitan monooleate, sorbitan sesquioleate, polyoxy ethylene hydrogenated castor oil 60.

Examples of suitable pH controllers include hydrochloric acid, sodium hydroxide, buffers and the like. Examples of suitable isotonic agents are glucose, D-sorbitol or D- mannitol, sodium chloride.

The administration of the agent may also be in the form of a composition containing a pharmaceutically acceptable carrier, diluent, excipient, suspending agent, lubricating agent, adjuvant, vehicle, delivery system, emulsifϊer, disintegrant, absorbent, preservative, surfactant, colorant, glidant, anti-adherent, binder, flavorant or sweetener, taking into account the physical, chemical and microbiological properties of the agent being administered.

For these purposes, the composition may be administered orally, parenterally, by inhalation spray, adsorption, absorption, topically, rectally, nasally, bucally, vaginally, intraventricularly, via an implanted reservoir in dosage formulations containing conventional non-toxic pharmaceutically-acceptable carriers, or by any other convenient dosage form. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, and intracranial injection or infusion techniques.

When administered parenterally, the composition will normally be in a unit dosage, sterile, pyrogen free injectable form (solution, suspension or emulsion, which may have been reconstituted prior to use) which is usually isotonic with the blood of the recipient with a pharmaceutically acceptable carrier. Examples of such sterile injectable forms are sterile injectable aqueous or oleaginous suspensions. These suspensions may be formulated according to techniques known in the art using suitable vehicles, dispersing or wetting agents and suspending agents. The sterile injectable forms may also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, for example, as solutions in 1,3-butanediol. Among the pharmaceutically acceptable vehicles and solvents that may be employed are water, ethanol, glycerol, saline, Ringer's solution, dextrose solution, isotonic sodium chloride solution, and Hanks' solution. In addition, sterile, fixed oils are conventionally employed as solvents or suspending mediums. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides, corn, cottonseed, peanut, and sesame oil. Fatty acids such as ethyl oleate, isopropyl myristate, and oleic acid and its glyceride derivatives, including olive oil and castor oil, especially in their polyoxyethylated versions, are useful in the preparation of injectables. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants.

The carrier may contain minor amounts of additives, such as substances that enhance solubility, isotonicity, and chemical stability, for example anti-oxidants, buffers and preservatives.

In addition, the composition may be in a form to be reconstituted prior to administration. Examples include lyophilization, spray drying and the like to produce a suitable solid form for reconstitution with a pharmaceutically acceptable solvent prior to administration.

Compositions may include one or more buffer, bulking agent, isotonic agent and cryoprotectant and lyoprotectant. Examples of excipients include, phosphate salts, citric acid, non-reducing sugars such as sucrose or trehalose, polyhydroxy alcohols, amino acids, methylamines, and lyotropic salts are preferred to the reducing sugars such as maltose or lactose.

When administered orally, the agent will usually be formulated into unit dosage forms such as tablets, caplets, cachets, powder, granules, beads, chewable lozenges, capsules, liquids, aqueous suspensions or solutions, or similar dosage forms, using conventional equipment and techniques known in the art. Such formulations typically include a solid, semisolid, or liquid carrier. Exemplary carriers include excipients such as lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, mineral oil, cocoa butter, oil of theobroma, alginates, tragacanth, gelatin, syrup, substituted cellulose ethers, polyoxyethylene sorbitan monolaurate, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and the like.

A tablet may be made by compressing or moulding the agent optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active, or dispersing agent. Moulded tablets may be made by moulding in a suitable machine, a mixture of the powdered active ingredient and a suitable carrier moistened with an inert liquid diluent.

The administration of the agent may also utilize controlled release technology.

For topical administration, the composition may be in the form of a solution, spray, lotion, cream (for example a non-ionic cream), gel, paste or ointment. Alternatively, the composition may be delivered via a liposome, nanosome, ribosome, or nutri-diffuser vehicle.

The preparation of pharmaceutical compositions is known in the art, for example Remington's Pharmaceutical Sciences, 18th ed., 1990, Mack Publishing Co., Easton, Pa. and U.S. Pharmacopeia: National Formulary, 1984, Mack Publishing Company, Easton, Pa.

Therapeutic delivery of biolomolecules is generally as described in Bladon, C. (2002) "Pharmaceutical Chemistry: Therapeutic Aspects of Biomolecules" John Wiley & Sons Ltd.

Viral and gene therapy techniques are as generally described in "Viral Vectors for Gene Therapy: Methods and Protocols" Edited by Jules G Constant, Curtis A Machida (2003) Humana Press Inc., "Gene Delivery to Mammalian Cells: Viral Gene Transfer Techniques" Edited by William C Heiser (2004) Humana Press Inc., "Viruses in Human Gene Therapy" Edited by J.H. Vos (1995) Carolina Academic Press, and "Viral Therapy Of Human Cancers" Edited by J.G. Sinkovics and J.C. Horwath (2005) Marcel Dekker.

The modulation of angiogenesis may be determined by a suitable method known in the art.

For example, inhibition of angiogenesis may be determined in xenograft model in nude mice, such as the method described by Holmquist-Mengelbier et al. (Cancer Cell 10(5):413-423, 2006). Corresponding vascularisation of the tumour can be scored via microscopy using standard histology. This is also generally accompanied with measurements of HIF-2 responsive genes in the tumours using techniques such as RNA protections assays on VEGF and EPO. Using this model allows determination whether reducing HIF-2 alpha levels with an inhibitor successfully reduces tumour growth in mice.

Alternatively, inhibition of angiogenesis may be determined by another method known in the art, such as the delayed appearance of neovascular structures, slowed development of neovascular structures, decreased occurrence of neovascular structures, slowed or decreased severity of angiogenesis-dependent disease effects, arrested angiogenic growth, or regression of previous angiogenic growth.

Other models for determination of the ability of an agent to inhibit angiogenesis include a mouse aortic explant model, chicken chorioallantoic membrane (CAM) assay or a corneal neovascularization model.

For example, the ability of an agent to inhibit angiogenesis in a chicken chorioallantoic membrane assay may be tested by contacting the chorioallantoic membrane with the agent applied to a methylcellulose disc. For the corneal neovascularization model, the agent may be applied as a topical composition containing the agent to the cornea, the cornea being scratched and inoculated with an agent to induce neovascularisation. Another method to study angiogenesis is the subcutaneous implantation of various artificial sponges (i.e. polyvinyl alcohol, gelatin) in animals. The agent to be evaluated may be injected directly into the sponges, which are placed in the centre of the sponge. Neovascularization of the sponges is assessed either histologically, morphometrically (vascular density), biochemically (haemoglobin content) or by measuring the blood flow rate in the vasculature of the sponge using a radioactive tracer.

As discussed above, numerous animal tumour models have also been developed to test the anti-angiogenic activity of test compounds. In many cases, tumour cells are engrafted subcutaneously and tumour size is determined at regular time intervals. Frequently used tumour cells include C6 rat glioma, B16BL6 melanoma, LLC, and Walker 256 carcinoma.

The present invention may also be used to screen candidate compounds as agents that can modulate the level and/or activity of a HIF-2 alpha protein, as agents that can modulate phosphorylation of a HIF-2 alpha protein, or as agents that can modulate angiogenesis.

In one embodiment, the present invention may be used to identify an agent that modulates the level and/or activity of a HIF-2 alpha protein, by identifying an agent that modulates phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.1) in a HIF-2 alpha protein.

Accordingly, in another embodiment the present invention provides a method of identifying an agent that modulates the level and/or activity of HIF-2 alpha protein, the method including identifying an agent that modulates phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in a HIF-2 alpha protein.

Methods for determining the level and/or activity of a HIF-2 alpha protein are known in the art and generally include for example immunological methods of detection of the protein, and assays measuring the ability of the protein to bind to DNA and/or transactivate transcription, which are known in the art. Methods for determining the extent of phosphorylation of a HIF-2 alpha protein are known in the art, as previously described herein.

Methods for screening agents involving the high-throughput screening of agents are specifically contemplated. For example, high throughput screening methods are as described in "High Throughput Screening" (2002) Humana Press Inc. edited by William P. Janzen.

In one embodiment, the method of identifying the agent is performed in a cell-free system.

In an alternative embodiment, the method of identifying the agent is performed in one or more cells, for example one or more cells in vitro, or one or more cells in a biological system.

The method may be used to identify an agent that reduces the level and/or activity of a HIF-2 alpha protein.

Accordingly, in one embodiment the present invention provides a method of identifying an agent that reduces the level and/or activity of HIF-2 alpha protein, the method including determining the ability of a candidate compound agent to reduce phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in a HIF-2 alpha protein, wherein a candidate compound that reduces phosphorylation is indicative of an agent that reduces the level and/or activity of HIF-2 alpha protein.

Alternatively, the method may be used to identify an agent that increases the level and/or activity of a HIF-2 alpha protein.

Accordingly, in another embodiment the present invention provides a method of identifying an agent that increases the level and/or activity of HIF-2 alpha protein, the method including determining the ability of a candidate compound to increase phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in a HIF-2 alpha protein, wherein a candidate compound that increases phosphorylation is indicative of an agent that increases the level and/or activity of HIF-

2 alpha protein.

In one embodiment, the candidate compound includes one or more of amino acid sequences selected from the group of amino acid sequences consisting of SEQ ID NOs.

3 to 25, or a functional variant thereof.

For example, the candidate compound may be a polypeptide including one or more of amino acid sequences selected from the group of amino acid sequences SEQ ID NOs. 3 to 25, or a functional variant thereof.

In one specific embodiment, the candidate compound is a polypeptide consisting of one of the amino acid sequences SEQ ID NOs. 3 to 25, or a functional variant thereof.

In another embodiment, the present invention may be used to identify an agent that modulates the level and/or activity of a HIF-2 alpha protein by screening candidate compounds including an amino acid sequence DFQLSPI (SEQ ID No.23) or a functional variant thereof.

Accordingly, in one embodiment the present invention provides a method of identifying an agent that modulates the level and/or activity of a HIF-2 alpha protein, the method including: providing a candidate compound including an amino acid sequence DFQLSPI

(SEQ ID NO.23), or a functional variant thereof; determining the ability of the candidate compound to modulate the level and/or activity of a HIF-2 alpha protein; and identifying the candidate compound as an agent that modulates the level and/or activity of a HIF-2 alpha protein.

The present invention may also be used to identify an agent that modulates phosphorylation of the specific serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in a HIF-2 alpha protein. Accordingly, in another embodiment the present invention provides a method of identifying an agent that modulates phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in a HIF-2 alpha protein, the method including: providing a candidate compound including an amino acid sequence DFQLSPI

(SEQ ID NO.23), or a functional variant thereof; determining the ability of the candidate compound to modulate phosphorylation of the serine residue; and identifying the candidate compound as an agent that modulates phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in a

HIF-2 alpha protein.

Methods for determining the extent of phosphorylation of a HIF-2 alpha protein are as described herein.

In another embodiment, the present invention may also be used to screen candidate compounds as being agents that modulate angiogenesis, by identifying an agent that modulates phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in a HIF-2 alpha protein.

Accordingly, in another embodiment the present invention provides a method of identifying an agent that modulates angiogenesis, the method including identifying an agent that modulates phosphorylation of the serine residue in the amino acid sequence FQLSPIC in a HIF-2 alpha protein.

The method may be used to identify an agent that inhibits or increases angiogenesis. Methods for determining the extent of angiogenesis in a biological system are known in the art, and are as previously described herein.

In one embodiment, the candidate compound includes one or more of amino acid sequences selected from the group of amino acid sequences SEQ ID NOs. 3 to 25, or a functional variant thereof. For example, the candidate compound may be a polypeptide including one or more of amino acid sequences selected from the group of amino acid sequences SEQ ID NOs. 3 to 23, or a functional variant thereof.

The present invention may also be used to identify an agent that modulates angiogenesis by screening candidate compounds including an amino acid sequence DFQLSPI (SEQ ID NO.23) or a functional variant thereof.

Accordingly, in one embodiment the present invention provides a method of identifying an agent that modulates angiogenesis, the method including: providing a candidate compound including an amino acid sequence DFQLSPI

In one embodiment, the candidate compound includes one or more of amino acid sequences selected from the group of amino acid sequences SEQ ID NOs. 3 to 25, or a functional variant thereof.

For example, the candidate compound may be a polypeptide including one or more of amino acid sequences selected from the group of amino acid sequences SEQ ID NOs. 3 to 23, or a functional variant thereof.

In one specific embodiment, the candidate compound is a polypeptide consisting of one of the amino acid sequences SEQ ID NOs. 3 to 25, or a functional variant thereof. The present invention also provides an agent identified according to the above described methods of identification, the use of the agents as therapeutic agents, and pharmaceutical compositions including such agents.

Finally, standard techniques may be used for recombinant DNA technology, oligonucleotide synthesis, and tissue culture and transfection (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y.

Description of Specific Embodiments

Reference will now be made to experiments that embody the above general principles of the present invention. However, it is to be understood that the following description is not to limit the generality of the above description.

Brief Description of the Figures

Figure 1 shows that HIF-2α is constitutively expressed in PC 12 cells and phosphorylated on S543 in the oxygen degradation domain. In (A) PC 12 cells were treated with vehicle DPP (100 μM) or MG132 (20 μM) for 6 h, cells harvested, and equal protein extracts subjected to SDS-PAGE and immunoblotting using anti-HIF-2α monoclonal antisera. In (B) the HIF-2α C-terminal ODDD was aligned using EclustalW across arrange of species. The conserved LAP domain, a potential sumoylation site, and S543 are highlighted. In (C), 293T cells were transfected with a cDNA encoding HA- tagged aa 486-630 of HIF-2α fused to a C-terminal GST tag. 24 h following transfection cells were labelled with 32P for 4 h, the GST fusion protein purified using affinity chromatography, and resulting samples subjected to SDS-PAGE prior to either autoradiography or immunob lotting using 12 CA5 antisera. In (D) and (E) the same protocol for (C) was repeated with the exception that during 32P labelling cells were incubated with the various kinase inhibitors at the concentration shown on the figure.

Figure 2 shows that HIF-2α phosphorylation on S543 confers stability to the HIF-2α oxygen degradation domain in PC 12 cells and instability during hypoxia. In (A) cDNA's encoding a S543A mutant, or wild-type N-terminal 6x Myc tagged HIF-2α ODDD's (aa 486-630) were stably transfected into PC12 cells. Cells were treated with vehicle (C, control) DPP (100 μM), MG 132 (20 μM), or DPP + MG 132 for 6 h. Cells were harvested, extracts prepared and equal protein samples subjected to SDS-PAGE and immunob lotting using (9E10 antisera). Position of the HIF-2α ODDD is shown on the figure. In (B) a stable line was generated using aa 486-549 of HIF-2α using the same N-terminal 6xMyc tag and directly compared with the other PC 12 recombinant lines using the same treatments as in (A). Positions of the 2 versions of the HIF-2α ODDD are as shown on the figure.

Figure 3 shows that during normoxia, S543 phosphorylation inhibits VHL-mediated ubiquitination of the HIF-2α oxygen degradation domain. In A(I) PC 12 cells were transfected with cDNA's encoding either HIF-2α ODDD or HIF-2α ODDD/S543A carrying an N-terminal 6xMyc tag and a cDNA encoding HA-tagged VHL-GST. Controls were transfected with VHL alone. 24 H following transfection cells were incubated with MG132 for 4 h, cell extracts prepared and expressed proteins subjected to affinity purification using GST resin. Input levels of HIF-2α ODDD from cell extracts and amounts purified with VHL-GST are as shown. In A(II) PC 12 transfections using HIF-2α ODDD cDNAs were repeated without VHL, cell extracts prepared following MG 132 incubation, and expressed proteins incubated with pre-purified VHL- GST derived from 293T cells. In (B) cDNA's encoding the mutant and wild-type HIF- 2α ODDD's were transfected into 293T cells together with a cDNA encoding HA- VHL-GST. 24 post-transfection cells were exposed to 6 h hypoxia, then rapid re- oxygenation to allow VHL capture of the HIF-2α ODDD. Ubiquitination assays were subsequently performed as previously described (Paltoglou, S., and Roberts, B. J. (2005) Oncogene 24(23): 3830-3835). In out levels of the HIF-2α ODDD are as shown. In (C) the dependence of multiubiquitination in this assay is demonstrated to depend on the availability of Ubc5.

Figure 4 shows that in the absence of VHL binding, S543 phosphorylation confers degradation of the HIF-2α oxygen degradation domain by the F-box protein, Fbw7. PC 12 or 293T cells were stably transfected with a P531A mutant of the HIF-2α ODDD cDNA described in Fig. 2. Cells were treated with vehicle or MG 132 (20 μM) for 6 h, cells harvested, extracts prepared, and samples subjected to SDS-PAGE and immunoblotting using 9E10 antisera. In (B) 293T cells stably expressing the P531A mutant as in (a) were transfected with cDNA's encoding either HA-VHL or HA-Fbw7. 24 h post-transfection cells were treated with vehicle or MG 132 for 4 h. Cells were harvested, extracts prepared and equal protein samples subjected to SDS-PAGE and immunoblotting with 9E10 antisera. This protocol enabled detection of the N-terminal 6xMyc tagged HIF-2α ODDD simultaneously with endogenous c-Myc. Blots were stripped and re-probed with 12CA5 antisera to detect HA-VHL and HA-Fbw7. In (C) a pull-down experiment was performed to detect whether the HIF-2α ODDD interacted with Fbw7. 293T cells were transfected with cDNA's encoding either the a P531A mutant or a P531A/S543A mutant, together with a cDNA encoding HA-Fbw7. 24 h post-transfection cells were treated with vehicle or MG 132 for 4 h and the HA-Fbw7 immunoprecipitated using 12CA5 antisera. Purified samples or input extracts were subjected to SDS-PAGE and immunoblotting using 9E10 antisera, followed by stripping and re-probing with 12CA5. Note the appearance of a slower migrating

Figure 5 shows the human C-terminal HIF-2α oxygen degradation domain.

Figure 6 shows in vitro phosphorylation of purified HIF-2α on S543 is inhibited by a peptide orthologue of the HIF-2α phosphorylation domain. In this figure HIF-2α ODDD was purified from HEK 293T cells and subjected to in vitro phosphorylation with increasing amounts of the peptide sequence shown using PC 12 cell extracts in the presence of ³²P ATP and ATP regenerating system. Samples were washed, electrophoresed and subjected to autoradiography. Control loading of the purified HIF- 2α is shown in the lower panel.

Figure 7 shows the effects of N-terminal myristoylated peptide analogue 15 (SEQ ID NO: 18) on phosphorylation of the HIF-2α oxygen degradation domain (ODDD) and induction of HIF-2α from human kidney cells (HEK293T).

Figure 8 shows the relationship between the observed tumour size vs peptide treatment (in μM) in the mouse Lewis Lung Carcinoma (LCC) model.

Example 1

Materials and Methods

cDNAs and sub-cloning- cDNAs used in this study were generously provided by the following labs. A cDNA encoding HA-tagged Fbw7 was obtained from Masaki Matsumoto (Kyushu University, Japan). A cDNA encoding HA-pVHL was originally obtained from W. Kaelin (Dana-Farber Cancer Institute). The cDNA encoding the HIF- ODDD used in this study was subcloned onto a 5' cDNA sequence encoding an N- terminal 6xMyc/6xHis tag as previously described (Lewis, M. D., and Roberts, B. J. (2004) Oncogene 23, 2315-2323). Generation of HA-tagged VHL-GST/EF-IRES- puromycin, has been described previously (Paltoglou, S., and Roberts, B. J. (2005) Oncogene 24(23): 3830-3835). A cDNA encoding an N-terminal HA-tagged ODDD of HIF-2α (aa 486-630) fused to a C-terminal GST tag was created using PCR.

Mutagenesis of cDNAs was performed using the Quickchange method. All cDNA's were sequenced following genetic manipulation.

Cell lines, antibodies and reagents-PC 12 cells were maintained in 10% horse serum/DMEM. All other cells were maintained in 10% FCS/DMEM. Stable cell lines were generated using either G418 or puromycin selection as previously described (Lewis, M.D. and Roberts B.J. (2003) Oncogene 22, 3992-7). Transient transfection was performed using Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions. 12CA5 antisera was obtained from Roche and 9E10 hybridoma from ATCC. HIF-2α and HIF- lα monoclonal antisera were obtained from Transduction Laboratories. CSN5 antisera was obtained from Biomol.

GST resin was obtained from Amersham Biosciences, Talon metal affinity resin from Becton-Dickinson. Protein-A sepharose was obtained from Sigma Chemicals. For hypoxia treatments, cells were incubated in purpose built air-tight containers in the presence of Oxoid anaerobic sachets.

MG 132 was obtained from Biomol.

Cell extract preparation and immunoblotting, radiolabeling, protein purification and bioinformatic analysis - CSN5 immunoprecipitation was performed by lysing 293T cells in whole cell extract (WCE) buffer (20 mM Tris-HCl pH 7.5, complete protease inhibitor (Roche Chemicals) 0.1 % Triton-X-100, and 0.4 M NaCl) followed by a 1 in 4 dilution in H₂O to reduce salt concentration. The extract was incubated for 4 h at 4⁰C and washed 3 times in WCE prior to the addition of SDS-PAGE sample buffer and immunoblotting. HA-Fbw7 immunoprecipitation using 12CA5 antisera was performed using the same procedure.

To radiolabel 293T cells with ³²P, approximately 200 uCi of ³²P orthophosphate (ICN Biomedicals) was added to 35 mm semi-confluent dishes in phosphate free DMEM (Invitrogen). Cells were incubated for 4 h, then washed with 1 ml PBS, followed by harvesting in ice cold WCE buffer supplemented with phosphatase inhibitor cocktail 1 (Sigma Chemicals).

Radiolabeled HIF-2α -ODDD-GST was purified using GST resin chromatography and purified samples subjected to SDS-PAGE and autoradiography, or, SDS-PAGE and immunoblotting with 12CA5 antisera to determine HIF-2α-ODDD levels. 6xMyc/6xHis tagged HIF-2α ODDD was purified from PC 12 cells using WCE buffer, with the NaCl concentration adjusted to 1.0 M prior to incubation with Talon resin for 3 h at 4⁰C. After 3 x 10 min washes in WCE buffer, samples were boiled in SDS-PAGE sample buffer prior to electrophoresis and immunoblotting. Protein alignments were performed using the EclustalW algorithm Example 2

HIF-2a is phosphorylated at a conserved serine residue under normoxic conditions by a

MAP kinase

Previous studies have shown variance in the hypoxic response between HIF- lα and HIF-2α, including altered duration of induction, induction in different cells, cytoplasmic retention of HIF-2α but not HIF- lα, and an emerging role for HIF- lα rather than HIF- 2α in much of the hypoxic response. Some reports have proposed that HIF-2α activates certain genes selectively such as EPO, others that HIF-2α is involved apoptosis, or in maintaining redox status. At least some of these differences are likely to account for the reported pro-tumorigenic activity of HIF-2α by comparison with HIF- lα, however, it is still unclear how the functions of HIF-I α and HIF-2α segregate at the molecular level.

During the course of our investigations into the inducibility of HIF- lα and HIF-2α across numerous cell lines we noted variable induction between the two in several cell types. Of particular interest was the phaeochromocytoma derived PC 12 cell line in which we noted little or no discernible increase in HIF-2α following hypoxia or DPP treatment for up to 6 h. This is in agreement with a previous study that showed under similar conditions a significant increase in HIF- lα expression.

Moreover, we were able to readily detect HIF-2α during normoxia in this cell type suggesting stabilization of the HIF-2α molecule during normoxia (Fig. IA). Though these changes could be explained by differences in proline hydroxylase selectivity, we investigated the oxygen degradation domain of HIF- lα and HIF-2α to determine regions of variance, particularly with respect to the VHL binding. Although the 2 regions are highly conserved, the C-terminal half of the VHL binding domain at S543 in the HIF-2α sequence differed from that of HIF- lα by forming part of a strong MAP kinase consensus sequence (Fig. IB). This serine is conserved amongst birds and mammals and is replaced by an asparagine in fish and amphibians. Extending the alignment of HIF- lα and HIF-2α further along the C-terminal of the oxygen degradation domain reveals little homology between the two suggesting some measure of structural or functional divergence beyond the critical VHL interaction. We next mutated S543 in the HIF-2α ODDD to determine whether it was phosphorylated in vivo in HEK 293T cells. Purifying the HIF-2α ODDD following ³²P labelling in these cells revealed a robust incorporation into the wild-type ODDD that was largely abolished in the S543A mutant (Fig. 1C). Since it is unlikely the alanine mutation has any effect on HIF-2α structure per se, we concluded that HIF-2α is constitutively modified on S543 in this cell type.

We next used a series of inhibitors to establish which kinase was responsible for this modification (Fig. ID and IE). From these data we were unable to detect any inhibition with the MAP kinase inhibitor PD-98059 and only slight inhibition using the PI3K inhibitor wortmannin. By contrast, phosphorylation was blocked by the p38 MAP kinase inhibitor SB-203580 between 10-20 uM, suggesting one, or perhaps several, of the stress-activated kinases belonging to this family is predominantly responsible for S543 phosphorylation in these cells. Wortmannin, it should be noted, has been reported to exhibit some inhibition towards p38 MAP kinases also, though it is impossible to rule out an indirect role for the PI3 kinase exerting a positive effect on p38 MAP kinase activity.

Example 3

S543 phosphorylation stabilizes the HIF-2a ODDD against degradation under normoxia and targets the HIF-2a ODDD for degradation under hypoxia

Within the context of this modification we next undertook experiments to determine its role on the regulation of the HIF-2α ODDD in PC 12 cells. We generated stable cell lines expressing either the HIF-2α ODDD or corresponding S543A mutant whereupon quite striking differences were observed. During normoxia the HIF-2α ODDD was clearly detectable whereas the S543A mutant was not (Fig. 2A), suggesting that S543 phosphorylation stabilizes the HIF-2α ODDD against VHL-dependent degradation.

Surprisingly, this same modification also targeted the HIF-2α ODDD for degradation during DPP treatment, a feature which was entirely absent in the S543A mutant (Fig. 2D). The net result is that the phosphorylated HIF-2α ODDD is only weakly inducible by DPP, but is stabilized by proteasome inhibitors and is therefore considered to be targeted to another degradation pathway unlike the S543A mutant. This effect appeared contingent on an intact region between aa 550-630 of the ODDD (Fig. 2B).

Overall, we contend that a high level of phosphorylation of HIF-2α on S543 can account for the regulation of endogenous HIF-2α observed in PC12 cells in Fig. IA. Identical findings were obtained for hypoxic treatment up to 6 h treatment, thus, this modification appears to work against the conventional rapid hypoxic activation of HIF- 2α observed in prior studies but nonetheless facilitates the expression of HIF-2α during normoxia.

Example 4

Phosphorylation ofS543 inhibits VHL mediated ubiquitination

To determine whether the molecular mechanism of HIF-2α stabilization involved a phosphorylation-induced inhibition of proline hydroxylase activity, we expressed the HIF-2α ODDD and corresponding S543A mutant in PC 12 cells and purified each protein with either co-expressed VHL E3 ubiquitin ligase or VHL E3 ligase purified separately from 293T cells (Paltoglou, S., and Roberts, B. J. (2005) Oncogene 24(23): 3830-3835).

In the presence of proteasome inhibitor the phosphorylated form of the HIF-2α ODDD exhibited increased binding to VHL compared to the S543A mutant (Fig. 3A), suggesting S543 phosphorylation does not inhibit proline hydroxylation.

These findings prompted us to investigate whether VHL-mediated degradation itself was impaired by S543 phosphorylation. When each of these forms was transfected into 293T cells, captured using VHL and subjected to ubiquitination in vitro it was apparent that the S543A mutant was efficiently ubiquitinated by comparison with the phosphorylated form (Fig. 3B). From these findings we propose that S543 phosphorylation hinders access of the E2 Ubc5 enzyme to lysine acceptors, or alternatively, blocks Ubc5 recruitment to the Cul2/Rbxl binding region. Both mechanisms are likely to involve conformational changes or steric hindrance provided by aa 540-630 of the HIF-2α ODDD.

Example 5

Fbw7 transfected into 293T cells restores HIF-2a ODDD degradation

A surprising finding from our studies using PC 12 cells was the instability of the S543 phosphorylated HIF-2α ODDD during hypoxia. To determine whether this degradation pathway was conserved in 293T cells we expressed a P531A mutant HIF-2α ODDD in both cell types and assessed its stability (Fig. 4A). In 293T cells the P531A mutant was relatively resistant to degradation, however, in PC 12 cells at similar molar expression levels the same protein was undetectable. Since 293T cells possess the ability to efficiently phosphorylate S543 another enzyme pathway must be present in PC 12 cells that accounts for this degradation.

Although many F-box and non F-box ubiquitin ligases target phosphoproteins for degradation the number that are expressed selectively in the CNS and not kidney is significantly restricted. For this reason we focused our investigation on the F-box protein Fbw7. This protein is expressed at high levels in the brain and heart, and has been shown to target cyclin E and c-Myc for phosphorylation dependent degradation. When the tagged version of this F-box protein was transfected into 293T cells stably expressing HIF-2α ODDD/P531A, levels of the ODDD were lowered to the same extent as the previously identified Fbw7 ubiquitin target c-Myc (Fig. 4B). This loss was prevented by proteasome inhibitor. Binding assays performed to assess the affinity of Fbw7 for the HIF-2α ODDD/P531A revealed that S543 phosphorylation was not required for the interaction but nonetheless conferred stability on the HIF-2α ODDD (Fig. 4C). Thus, the minimal phosphodegron for Fbw7 binding within the HIF-2α ODDD awaits further characterization.

Interestingly, we consistently observed a slower migrating band upon incubation of the S543A mutant with MG 132, suggesting there may be other post-translational modifications to the HIF-2α ODDD that accumulate when degradation is blocked. One, or several of these could be incorporated into a HIF-2α phosphodegron. Importantly, we do not rule out the contribution of other E3 ligases to HIF-2α degradation in PC 12 cells. Merely, that in the model system shown F-box proteins that use phosphodegrons as their primary recognition motif are capable of degrading phosphorylated HIF-2α in the absence of VHL binding.

There is a growing body of literature that supports a role for HIF-2α in non-hypoxic gene regulation. In the present study stress-activated p38 MAP kinases appear to contribute to HIF-2α stabilization during normoxia and to promote destruction in PC 12 cells during hypoxia. Perhaps this is one mechanism whereby the selective induction of HIF-lα-inducible genes is maintained in certain tissues during low oxygen tension, where HIF- lα and HIF-2α presumably compete for a limiting pool of coactivator. An alternative, although not mutually exclusive scenario, is that S543 phosphorylation allows HIF-responsive gene transcription to be regulated by multiple pathways that converge on p38 MAP kinase activity. Such a scenario would support HIF-2α as a "stress-response" factor. With reference to normal tissue, it is unclear form our work whether changes in HIF-2α expression signify an inherent feature of transformed lines, in which one might anticipate cellular stress and thereby p38 MAP kinase activity to be constitutively high.

Example 6

A peptide based on the S543 phosphorylation site inhibits HIF-2 a phosphorylation

The human C-terminal HIF-2α oxygen degradation domain is shown in Figure 5.

The crystal structure of VHL bound to a HIF peptide suggests the ODDD domain of HIF-lα/2α is not rigid in the vicinity of the VHL binding site. Possibly for this reason the HIF- ODDD has thus far resisted structure analysis. This lack of structure in the ODDD may be one reason it is efficiently phosphorylated, a feature which may render it an excellent target for peptide orthologue inhibitors.

In order to test which peptide best inhibits the phosphorylation of S543, a battery of peptides will be generated to determine the optimal inhibitory sequence. At first instance, 20 peptides will be screened for their inhibitory activity towards HIF- 2α S543 phosphorylation. Their selection is based on the sequence surrounding S543 on HIF-2α and their hydrophilicity/solubility:

MDGEDFQLSPICPEERLLAE (SEQ ID NO. 3) MDGEDFQLSPICPEERLLA (SEQ ID NO. 4) MDGEDFQLSPICPEERLL (SEQ ID NO. 5) MDGEDFQLSPICPEERL (SEQ ID NO. 6) MDGEDFQLSPICPEER (SEQ ID NO. 7) MDGEDFQLSPICPEE (SEQ ID NO. 8) MDGEDFQLSPICPE (SEQ ID NO. 9) MDGEDFQLSPICP (SEQ ID NO. 10) MDGEDFQLSPIC (SEQ ID NO. 11) EDFQLSPICPEER (SEQ ID NO. 12) EDFQLSPICPEE (SEQ ID NO. 13) EDFQLSPICPE (SEQ ID NO. 14) DFQLSPICPE (SEQ ID NO. 15) EDFQLSPICPEERLLAEN (SEQ ID NO. 16) YIPMDGEDFQLSPIC (SEQ ID NO. 17) IPMDGEDFQLSPICPEER (SEQ ID NO. 18) PMDGEDFQLSPICPEER (SEQ ID NO. 19) YIPMDGEDFQLSPICPEER (SEQ ID NO. 20) IPMDGEDFQLSPICPEER (SEQ ID NO. 21) PMDGEDFQLSPICPEER (SEQ ID NO. 22)

Example 7

Identification of a peptide inhibitor ofS543 phosphorylation ofHIF-2a

A peptide orthologue of the HIF-2α consensus phosphorylation site defined by SEQ ID NO. 12 has been tested in the assay system.

In this assay, a transiently expressed cDNA construct expressing the GST-tagged oxygen degradation domain of HIF-2α was purified from human cells using GST affinity chromatography and incubated with 200 μg of extract from PC 12 (phaeochromocytoma) cells prepared by hypotonic lyis in a buffer containing 10 mM Tris-HCl pH 7.5, 5 mM PMSF, 1 mM MgCl₂ and complete protease inhibitor tablet (Roche). Radiolabeling of the HIF-2α was accomplished by incubating the sample with ATP regenerating system and approximately 10 μCi of ³²P ATP for 1 h at 37°C. Inhibitory peptides were added at different concentrations 15 min before the addition of radiolabel. Following labeling, HIF-2α was re -purified using GST chromatography, SDS-PAGE sample buffer added and radiolabeled HIF-2α and HIF-2α levels determine by autoradiography and immunoblotting respectively.

As shown in Figure 6, the results of this assay confirm the ability of such assays as screening methods to identify agents that can modulate phosphorylation of a HIF-2 alpha protein, and thereby modulate the level and/or activity of a HIF-2 alpha protein.

Furthermore, the assay also indicates that the peptide defined by SEQ ID NO: 12 can modulate phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in the HIF-2 alpha protein.

Additional peptides were also screened to generate candidates suitable from in- vitro inhibition studies for testing in-vivo using N-terminal myristoylation (to facilitate transport across the cell membrane). These peptides are listed below:

MDGEDFQLSPICPEERLLAE (SEQ ID NO. 3) MDGEDFOLSPICPEERLLA (SEO ID NO. 4) MDGEDFOLSPICPEERLL (SEO ID NO. 5) MDGEDFOLSPICPEERL (SEO ID NO. 6) MDGEDFOLSPICPEER (SEO ID NO. 7) MDGEDFQLSPICPEE (SEQ ID NO. 8) MDGEDFQLSPICPE (SEQ ID NO. 9) MDGEDFQLSPICP (SEQ ID NO. 10) MDGEDFQLSPIC (SEQ ID NO. 11) EDFQLSPICPEER (SEQ ID NO. 12) EDFQLSPICPEE (SEQ ID NO. 13) EDFQLSPICPE (SEQ ID NO. 14) DFQLSPICPE (SEQ ID NO. 15) EDFQLSPICPEERLLAEN (SEQ ID NO. 16) YIPMDGEDFQLSPIC (SEQ ID NO. 17) IPMDGEDFOLSPICPEER (SEO ID NO. 18) PMDGEDFQLSPICPEER (SEQ ID NO. 19) YIPMDGEDFQLSPICPEER (SEQ ID NO. 20) IPMDGEDFQLSPICPEER (SEQ ID NO. 21) PMDGEDFQLSPICPEER (SEQ ID NO. 22)

The underlined peptides demonstrated inhibition at micoromolar concentrations.

The peptides defined by SEQ ID NO: 6, 7 and 18 were selected for myristoylation due to their reproducibility in inhibiting S543 phosphorylation in-vitro.

Example 8

Screening cell permeable peptide inhibitors of S543 phosphorylation on HIF-2a.

From a screen of peptides that were tested for in vitro inhibition of HIF-2α phosphorylation on S543, one was selected for N-terminal myristoylation to facilitate permeability across living human cells.

This peptide (SEQ ID NO: 18) was tested to determine whether it inhibited phosphorylation of S543 from an expressed HIF-2α ODDD in-vivo.

At a concentration between 50 and 100 micromolar the peptide moderately inhibited phosphorylation at normoxia (7A). It should be pointed out that these conditions do not simulate high endogenous phosphorylation levels of the HIF-2α that we have observed in PC 12 cells. In part, this is due to the fact that p38 MAP kinases are activated by stress conditions such as inflammation and mild hypoxia. We believe these conditions are found in tumours that express HIF-2α. Therefore, we tested endogenous HIF-2α expression in human kidney cells at 5% oxygen, a mildly hypoxic condition known to activate stress kinases (7B). Under these conditions the inhibitor significantly blocked the induction/expression of human HIF-2α.

Example 9

Screening of candidate compounds as inhibitors of angiogenesis

Neuroblastoma xenografts in nude athymic mice may be established using the method essentially as described in Holmquist-Mengelbier et al. (2006) Cancer Cell 10(5):413- 423.

Candidate anti-angiogenic agents may be tested for example by subcutaneous delivery, intraperitoneal injection, or direct injection into tunours. A suitable concentration of the agent ranges from 0.1 -100 mg/kg/day. Inhibition of angiogenesis may be determined by assessment of the reduction in angiogenesis as compared to vehicle.

At first instance, the following peptides including the minimal sequence DFQLSPI (SEQ ID NO.23) will be screened for their ability to act as anti-angiogenic agents:

Example 10

Activity of myristoylated peptides tested in a mouse tumour model to determine anti- tumorigenic activity

Two peptides were chosen for myristoylation and use in the in-vivo tumour studies. The peptide sequences were as follows:

Myr-MDGEDFQLSPICPEER ('peptide R'; SEQ ID NO: 7)

Myr-MDGEDFQLSPICPEERL ('peptide RL'; SEQ ID NO: 6)

These peptides were selected based on their relative hydrophilicity (solubility) compared to some of the other peptides tested in-vitro. This decision was considered a pre-requisite to get them into the tumours. In addition, it was felt that this was a more important factor for animal studies than their inhibitory capacity in-vitro, since all of the peptides inhibited S543 phosphorylation in-vitro at higher > μM concentrations.

The N-terminal myristoylation was carried out to facilitate peptide entry across the lipid bilayer of the cell.

Three different tumour cell lines, Hep3B hepatocellular carcinoma, EL-4 thymic lymphoblastic lymphoma (EL-4) and Lewis Lung carcinoma (LLC), are widely used subcutaneous transplantable tumour models that are syngeneic (genetically identical) to C57BL/6 mice. The last cell line was chosen since it has been reported that these tumours are heavily macrophage infiltrated, which should reflect a certain degree of inflammation (a stimulator of p38 MAP kinase).

Since a) the mechanism of HIF-2α induction observed in our studies appears to involve an inflammation-responsive p38 MAP -kinase pathway, b) tumour infiltrated macrophages exhibit HIF-2α staining, and c) Lung carcinomas exhibit GLUTl expression, (a HIF-2α marker gene) it was proposed to repeatedly inject the mice with peptides to determine whether they would inhibit tumour growth and/or marker activities associated with HIF-2α activity.

Cell permeable peptides were prepared in a ammonium hydroxide solution for injection into mice in which tumours will be grown.

Five mice were used per group. Mice were inoculated with 10⁶ LLC cells using a 23G needle under the skin of one flank and tumours allowed to develop. Peptides were administered twice daily at 10 or 100 μM after tumours had been established to grow 4- 5 days following inoculation with tumour cells. After 7-10 further days of tumour growth mice were euthanized, tumours excised and weighed.

Figure 8 shows the relationship between the observed tumour size vs peptide treatment (in μM). As can be seen, peptide RL at 100 μM inhibited tumour growth.

Tumours from Vehicle and the two 100 μM peptide concentration groups were further analysed for HIF marker expression. Tumours were homogenized in 500 μL of an ice cold buffer containing 0.25 M KCl, 0.2% Tween-20, 10 mM Tri-HCl pH 8.0, and complete protease inhibitor (Roche). Samples were left on ice for 15 min, centrifuged at 13,000 x g for 10 min and the supernatant used for protein analysis. Protein content was estimated using the Pierce protein estimation kit. Equal samples were loaded on SDS- PAGE gels and blotted for GLUTl, tubulin, phospho-GSK-3beta, and HIF-2α expression. Phospho GSK-3beta is a general measure of tumorigenicity in many cell types but may not reflect a direct link to HIF-2α expression or activity. Blots were scanned and protein levels quantitated using NIH image. The results are shown below in Tables 1 and 2. Table 1 shows the levels of tubulin and GLUTl per mg of protein from tumours in the vehicle group and the peptide RL and R at 100 μM groups. Tubulin is an internal control and is a measure of overall intracellular protein content. Levls of tubulin and GLUTl are expressed as arbitrary densitometric units (ADU) per mg of protein. S. D. refers to standard deviation.

Table 1 : Tubulin and GLUTl per mg of protein

Treatment

Tubulin Tubulin S.D GLUTl GLUTl S.D. (n=4)

Vehicle 1.2 0.2 2.12 0.42

RL 100 uM 1.1 0.1 1.9 0.32

R lOO uM 1.05 0.22 1.8 0.2

Table 2 shows the levels of phospho GSK-3beta per mg of protein from tumours in the vehicle control group and the peptide RL at 100 uM group. HIF-2α was not detectable in these tumours, owing to its instability and/or low level of expression in the LLC tumour cells. Levels are expressed as arbitrary densitometric units (ADU).

Table 2: Phospho GSK-3beta per mg of protein

Neither peptide influenced GLUTl (the HIF-2α marker gene) per mg protein though it should be noted that the low tumour weight in the RL 100 μM treatment meant that total GLUTl levels in an absolute sense were also low. It is conceivable that GLUTl bears a direct relationship to tumour vascularisation and therefore weight. In other words, it is a growth marker and not to be compared using mg of protein per se.

Finally, it will be appreciated that various modifications and variations of the methods and compositions of the invention described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are apparent to those skilled in the art are intended to be within the scope of the present invention.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A method of modulating the level and/or activity of a HIF-2 alpha protein, the method including modulating phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in the HIF-2 alpha protein.

2. A method according to claim 1, wherein the HIF-2 alpha is present in a cell.

3. A method according to claim 2, wherein increasing phosphorylation of the serine residue increases the level and/or activity of the HIF-2 alpha protein in the cell under normoxic conditions.

4. A method according to claim 2, wherein decreasing phosphorylation of the serine decreases the level and/or activity of the HIF-2 alpha protein in the cell under normoxic conditions.

5. A method according to claim 2, wherein increasing phosphorylation of the serine residue decreases the level and/or activity of the HIF-2 alpha protein in the cell under hypoxic conditions.

6. A method according to claim 2, wherein decreasing phosphorylation of the serine residue increases the level and/or activity of the HIF-2 alpha protein in the cell under hypoxic conditions.

7. A method according to any one of claims 2 to 6, wherein the modulation of phosphorylation of the serine residue modulates degradation of the HIF-2 alpha protein in the cell.

8. A method according to any one of claims 2 to 7, wherein the modulation of phosphorylation of the serine residue includes modulating activity of a mitogen- activated kinase in the cell.

9. A method according to claim 8, wherein the mitogen- activated kinase is p38 MAP kinase.

10. A method according to any one of claims 2 to 9, wherein the modulation of the phosphorylation of the serine residue includes exposing the cell to an agent that modulates phosphorylation of the serine residue in the HIF-2 alpha protein.

11. A method according to claim 10, wherein the agent includes the amino acid sequence DFQLSPI (SEQ ID NO.23), or a functional variant thereof.

12. A method according to any one of claims 2 to 11, wherein the HIF-2 alpha protein includes endogenous HIF-2 alpha protein present in the cell.

13. A method according to any one of claims 2 to 12, wherein the cell is a cell in a biological system.

14. A method according to claim 13, wherein the biological system is a human or animal subject.

15. A method according to claim 14, wherein the human or animal subject is a subject susceptible to, or suffering from, undesired and/or uncontrolled angiogenesis.

16. A method according to claim 15, wherein the subject is susceptible to, or suffering from one or more of cancer; angiogenesis associated with solid tumours; angiofibroma; corneal neovascularisation; retinal/choroidal neovascularization; arteriovenous malformations; arthritis, including rheumatoid arthritis, lupus and other connective tissue disorders; Osier- Weber syndrome; atherosclerotic plaques; psoriasis; pyogenic granuloma; retrolental fibroplasias; scleroderma; granulations, hemangioma; trachoma; hemophilic joints; vascular adhesions and hypertrophic scars; diseases associated with chronic inflammation including sarcoidosis and inflammatory bowel diseases such as Crohn's disease and ulcerative colitis.

17. A method of modulating angiogenesis in a biological system, the method including modulating phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in a HIF-2 alpha protein in one or more cells in the biological system.

18. A method according to claim 17, wherein angiogenesis is inhibited in the biological system.

19. A method according to claim 18, wherein the angiogenesis is associated with one or more the following diseases, conditions or states: cancer; growth of a solid tumour; angiofibroma; corneal neovascularisation; retinal/choroidal neovascularization; arteriovenous malformations; arthritis, including rheumatoid arthritis, lupus and other connective tissue disorders; Osier- Weber syndrome; atherosclerotic plaques; psoriasis; pyogenic granuloma; retrolental fibroplasias; scleroderma; granulations, hemangioma; trachoma; hemophilic joints; vascular adhesions and hypertrophic scars; diseases associated with chronic inflammation including sarcoidosis and inflammatory bowel diseases such as Crohn's disease and ulcerative colitis.

20. A method according to claim 17, wherein the angiogenesis is promoted in the biological system.

21. A method according to any one of claims 17 to 20, wherein the modulation of the phosphorylation of the serine residue includes exposing one or more cells in the biological system to an agent that modulates phosphorylation of the serine residue in the HIF-2 alpha protein.

22. A method according to claim 21, wherein the agent includes the amino acid sequence DFQLSPI (SEQ ID NO.23), or a functional variant thereof.

23. A method of preventing and/or treating a disease, condition or state associated with undesired and/or uncontrolled angiogenesis in a subject, the method including administering to the subject an agent that modulates phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in a HIF-2 alpha protein.

24. A method according to claim 23, wherein the agent includes an amino acid sequence DFQLSPI (SEQ ID NO.23), or a functional variant thereof.

25. A method according to claims 23 or 24, wherein the disease, condition or state is one or more the following diseases, conditions or states: cancer; growth of a solid tumour; angiofibroma; corneal neovascularisation; retinal/choroidal neovascularization; arteriovenous malformations; arthritis, including rheumatoid arthritis, lupus and other connective tissue disorders; Osier- Weber syndrome; atherosclerotic plaques; psoriasis; pyogenic granuloma; retrolental fibroplasias; scleroderma; granulations, hemangioma; trachoma; hemophilic joints; vascular adhesions and hypertrophic scars; diseases associated with chronic inflammation including sarcoidosis and inflammatory bowel diseases such as Crohn's disease and ulcerative colitis.

26. Use of an agent that modulates phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in a HIF-2 alpha protein in the preparation of a medicament for modulating angiogenesis.

27. Use of an agent according to claim 26, wherein the agent includes an amino acid sequence DFQLSPI (SEQ ID NO.23), or a functional variant thereof.

28. An agent that modulates the level and/or activity of a HIF-2 alpha protein by modulating phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in a HIF-2 alpha protein.

29. An agent according to claim 28, wherein the agent includes an amino acid sequence DFQLSPI (SEQ ID NO.23), or a functional variant thereof.

30. An agent according to claim 29, wherein the agent is a polypeptide consisting of an amino acid sequence selected from the group of SEQ ID Nos 3 to 25, or a functional variant thereof.

31. An agent that modulates phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in a HIF-2 alpha protein, the agent including an amino acid sequence DFQLSPI (SEQ ID NO.23), or a functional variant thereof.

32. An agent according to claim 31, wherein the agent includes an amino acid sequence DFQLSPI (SEQ ID NO.23), or a functional variant thereof.

33. An agent according to claim 32, wherein the agent is a polypeptide consisting of an amino acid sequence selected from the group of SEQ ID Nos 3 to 25, or a functional variant thereof.

34. An agent that modulates angiogenesis, the agent modulating phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in a HIF-2 alpha protein.

35. An agent according to claim 34, wherein the agent is an anti- angiogenic agent.

36. An agent according to claim 34, wherein the agent is a pro-angiogenic agent.

37. An agent according to any one of claims 34 to 36, wherein the agent includes an amino acid sequence DFQLSPI (SEQ ID NO.23), or a functional variant thereof.

38. An agent according to claim 37, wherein the agent is a polypeptide consisting of an amino acid sequence selected from the group of SEQ ID Nos 3 to 25, or a functional variant thereof.

39. An agent that modulates angiogenesis, the agent including an amino acid sequence DFQLSPI (SEQ ID NO.23), or a functional variant thereof.

40. An agent according to claim 39, wherein the agent is a polypeptide consisting of an amino acid sequence selected from the group of SEQ ID Nos 3 to 25.

41. An agent according to claims 39 or 40, wherein the agent is an anti- angiogenic agent.

42. An agent according to claims 39 or 40, wherein the agent is a pro-angiogenic agent.

43. A pharmaceutical composition including an agent according to any one of claims 28 to 42.

44. An isolated polypeptide consisting of an amino acid sequence selected from the group of amino acid sequences as provided by SEQ ID Nos 3 to 25, or a functional variant thereof.

45. A method of preventing and/or treating a disease, condition or state associated with undesired and/or uncontrolled angiogenesis in a subject, the method including administering to the subject an effective amount of a polypeptide according to claim 44.

46. Use of a polypeptide according to claim 44 in the preparation of a medicament.

47. Use according to claim 46, wherein the medicament is used for inhibiting angiogenesis.

48. A method of identifying an agent that modulates the level and/or activity of HIF-2 alpha protein, the method including identifying an agent that modulates phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in a HIF-2 alpha protein.

49. A method of identifying an agent that modulates angiogenesis, the method including identifying an agent that modulates phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in a HIF-2 alpha protein.

50. A method of identifying an agent that modulates the level and/or activity of a HIF-2 alpha protein, the method including: providing a candidate compound including an amino acid sequence DFQLSPI

(SEQ ID NO.23), or a functional variant thereof; determining the ability of the candidate compound to modulate the level and/or activity of a HIF-2 alpha protein in a cell; and identifying the candidate compound as an agent that modulates the level and/or activity of a HIF-2 alpha protein in a cell.

51. A method of identifying an agent that modulates angiogenesis, the method including: providing a candidate compound including an amino acid sequence DFQLSPI

52. A method of identifying an agent that modulates phosphorylation of the serine residue in the amino acid sequence FQLSPIC (SEQ ID NO.l) in a HIF-2 alpha protein, the method including: providing a candidate compound including an amino acid sequence DFQLSPI

HIF-2 alpha protein.

53. An agent identified according to the method of any one of claims 48 to 52.