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WO2005060996A2 - Method of altering the pka type i signalling pathway - Google Patents

Method of altering the pka type i signalling pathway Download PDF

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Publication number
WO2005060996A2
WO2005060996A2 PCT/GB2004/005396 GB2004005396W WO2005060996A2 WO 2005060996 A2 WO2005060996 A2 WO 2005060996A2 GB 2004005396 W GB2004005396 W GB 2004005396W WO 2005060996 A2 WO2005060996 A2 WO 2005060996A2
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Prior art keywords
binding
inhibitor
sequence
seq
ezrin
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PCT/GB2004/005396
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French (fr)
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WO2005060996A3 (en
Inventor
Kjetil TASKÉN
Anja Ruppelt
Mikaela GRÖNHOLM
Einar Martin Aandahl
Derek Tobin
Cathrine Carlson
Olli CARPÉN
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Lauras As
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Priority claimed from GB0329869A external-priority patent/GB0329869D0/en
Application filed by Lauras As filed Critical Lauras As
Publication of WO2005060996A2 publication Critical patent/WO2005060996A2/en
Publication of WO2005060996A3 publication Critical patent/WO2005060996A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)

Definitions

  • the present invention relates to inhibitors which abolish the function of cAMP dependent protein kinase (PKA) , type I and their use to produce pharmaceutical preparations to treat or prevent immunosuppressive diseases. More specifically, the present invention provides inhibitors of binding between specific binding partners of the PKA type I complex involved in PKA type I signalling. In particular inhibitors of the binding between ezrin and PKA type I, ezrin and EBP50 and EBP50 and Cbp/PAG are provided.
  • the immune system of mammals has evolved different strategies to defend the organism against ,the variety of potentially * infectious agents. The ability to acquire specific and anamnestic responses against intruders relies on the adaptive immune system.
  • the main players in the adaptive immune system are B and T lymphocytes, and the specific recognition of antigen by these cells is mediated by receptors with some degree of structural similarity, yet which are functionally very different.
  • the different receptor specificities are made possible through somatic rearrangement of a limited number of genes and are clonally distributed.
  • the main strategy of this system is to generate a nearly unlimited number of specificities to cover the recognition of almost any foreign antigen.
  • Immunological memory is partly a result of clonal expansion of subsets of T and B cells reacting with a particular antigen, and enables the organism to respond more quickly at the second encounter with the same antigen.
  • Cell proliferation is used as a marker of immune activation.
  • Cyclic AMP-dependent protein kinase is an enzyme present in all cells. Hormones and neurotransmitters binding to specific receptors stimulate the generation of the second messenger 3 ' , 5 ' - cyclic adenosine monophosphate (cAMP) . Cyclic AMP is one of the most common and versatile second messengers. The best characterized and major downstream effector mechanism whereby cAMP exerts its effects involves binding to and activating PKA. PKA is a serine/threonine protein kinase which phosphorylates a number of different proteins within the cell, and thereby regulates their activity.
  • PKA PKA regulates a vast variety of cellular processes such as metabolism, proliferation, differentiation and regulation of gene transcription.
  • the great diversity of cellular processes mediated by cAMP and PKA strongly suggests that there exists mechanisms that provide the required sensitivity and specificity of the effector pathway to ensure that rapid and precise signalling processes take place.
  • Specificity can be achieved by tissue- and cell-type specific expression of PKA isoforms with different biochemical properties.
  • targeting of PKA isoforms by A- kinase anchoring proteins (AKAPs) provides a higher level of specificity to the signalling process by localizing PKA to defined subcellular sites in close proximity to the substrate.
  • Anchoring of PKA by AKAPs may also tune the sensitivity of the signal pathway by recruiting PKA into multiprotein complexes that include phosphodiesterases and protein phosphatases as well as other signal proteins in addition to PKA (Michel and Scott, 2002, Ann. Rev. Pharmacol. Toxicol . , 42, p235- 257) .
  • PKA is made up of four different subunits, a regulatory (R) subunit dimer and two catalytic (C) subunits.
  • R regulatory
  • C catalytic
  • two main classes of PKA isozymes, PKA type I and PKA type II (PKAI and PKAII, respectively) have been described.
  • PKAI and PKAII can be distinguished by their R subunits, designated RI and RII.
  • Isoforms of RI and RII are referred to as Rio * , Rl ⁇ , Rll ⁇ and Rll ⁇ .
  • the C subunits also exist as isoforms referred to as Co., C ⁇ and Cy.
  • the different subunits may form multiple forms of PKA (isozymes) with potentially more than 18 different forms .
  • Activation occurs upon binding of cAMP to the R subunits followed by the release of the active catalytic subunit.
  • PKA type II is mainly particulate and associated with AKAPs whereas PKA type I is both soluble and particulate although PKA type I anchoring has remained more elusive.
  • PKA type I is recruited to the lipid raft fraction of the cell membrane upon T- cell activation, and colocalizes with the TCR-CD3 complex (Skalhegg et al. , 1994, Science, 263, p84-87) .
  • Lipid rafts are specialised membrane domains enriched in certain lipids, cholesterol and proteins. Their primary function is believed to be in signalling transduction. In particular they are considered to be the site of recruitment for various signalling molecules crucial for T cell activation.
  • PKA is a key negative regulator of lymphocyte function. The present inventors and others have shown that cAMP inhibits T lymphocyte proliferation induced through the T cell antigen receptor/CD3 complex (TCR/CD3) .
  • T cells express both PKAI and PKAII. However, only the selective activation of PKAI is sufficient to mediate the inhibitory effect of cAMP. In addition, we have demonstrated that PKAI, but not PKAII, redistributes to, colocalizes with and inhibits signalling through antigen receptors on T and B cells and natural killer cells and regulates mitogenic responses in T and B cells and acute cytotoxic responses in NK cells.
  • PKA type I mediates an inhibitory effect on the T cell activation cascade that involves activation of C- terminal Src kinase (Csk) by phosphorylating residue S364 (Vang et al, 2001, J. Exp. Med., 193, p497-507) .
  • PKAI serves as a key negative regulator of lymphocyte functions, e.g. mitogenic and cytotoxic responses initiated through antigen receptors.
  • the inventors' work has led to the conclusion that modulation of normal immune responsiveness by activation of PKA type I is a negative feedback mechanism. Dysregulation of this system may lead to immunological overshoot or impaired immune functions .
  • HIV Human immunodeficiency virus
  • CVI common variable immunodeficiency
  • T cell dysfunction is the immunological hallmark of HIV infection.
  • Defective lymphocyte cytokine production and impaired proliferative responses on stimulation are early signs of immunodeficiency in these patients, manifested even before a decline in CD4+ lymphocytes counts is observed.
  • B cell dysfunction with impaired antibody synthesis is the major immunological characteristic of CVI patients.
  • the immunological abnormalities in CVI are not restricted to B cells, but often also involve T cell dysfunction, e.g. impaired proliferative responses on stimulation.
  • the B cells in CVI patients are not necessarily intrinsically defective, and impaired T cell "help" may be of importance for the B cell defects in these patients. T cell dysfunction may also be of importance for certain clinical manifestations in these patients not necessarily related to defective antibody production, e.g. increased incidence of granulomata and malignancies.
  • antiretroviral therapy is the main component in the treatment of HIV- infected patients.
  • potent antiretroviral combination therapy may markedly increase the CD4+ and CD8+ lymphocytes counts in HIV-infected patients, impaired T cell function seems to persist, as indicated in the observations made in Example 1, table I and 2B of WO98/48807.
  • Immunoglobulin substitution is the main component in the treatment of CVI patients.
  • this substitution therapy does not restore the defective T and B cell function.
  • some clinical complications e.g. noncaseating granulomata and persistent viral infections, there is a need for therapy which may more directly enhance T cell function.
  • impaired T cell function is a well recognized immunological feature of both HIV infection and CVI, the exact molecular mechanism for this T cell impairment was not known.
  • Therapeutic modalities directed against such intracellular defects are expected to be of major importance in the treatment of these patients and may have the potential to restore important immunological defects in HIV-infected patients and in patients with CVI.
  • protein kinase A type I redistributes to and colocalizes with the antigen receptor complex upon activation and capping of T and B cells (Skalhegg et al , 1994, supra) .
  • This anchoring of protein kinase A type I supports a role for this specific isozyme in modulation of immune responses mediated through receptors on lymphoid cells.
  • discoveries by the inventors and as described in WO98/48809 show that there is an increased activation of protein kinase A type I in T cells from patients with HIV infection or CVI.
  • the inventors further demonstrated that activation of this isozyme of PKA leads to inhibition of immune function that can be reversed by selectively inhibiting the type I and not the type II isozymes of PKA.
  • the inventors sought compounds aimed at reversing the inappropriate activation of protein kinase A type I in immunodeficiencies (such as HIV, CVI) and thereby restoring T cell function and immune responsiveness.
  • the present inventors therefore set about developing strategies to specifically increase T cell immune function and reverse T cell dysfunction in human immunodeficiency virus infected and common variable immunodeficiency patients by using suitable compounds interfering with the cAMP/PKA pathway in T cells.
  • PKA type I as an immune modulator is shared among all lymphoid cells (T cells, B cells, NK cells) disruption of the cAMP/PKA pathway is also expected to be relevant to B and NK cell function.
  • Appropriate mechanisms for improving T cell function as developed by the inventors are described in WO98/48809, which is incorporated herein by reference.
  • the strategies relied on disruption of the cAMP-induced inhibition of T cell immune responses by abolishing PKA type I/RIoc signalling. Specifically, a number of specific mechanisms for disrupting the effects mediated by cAMP dependent protein kinase and hence stimulation of immune function were described.
  • protein kinase A type I function was inhibited by removal of the activated enzyme from substrates in the antigen receptor complex that are relevant for inhibition of immune function in T cells.
  • the search for new and improved modulators of PKA type I signalling and hence of immune function continues.
  • the inventors have now identified the involvement of a number of molecules not previously known to be involved in signalling via PKA type I which thus constitute attractive targets to mediate cell signalling.
  • the inventors have now identified a new AKAP which recruits and binds PKA type I and is hence involved in PKA type I signalling. This molecule's involvement in the signalling pathway provides a number of molecules which may be targeted to intervene in the signalling process.
  • Ezrin is an AKAP responsible for anchoring PKA type I to the TCR-CD3 complex during T cell activation and capping.
  • Ezrin also binds to EBP50 (ERM-binding phosphoprotein of 50kDa) which in turn binds to Cbp/PAG (Csk-binding protein or phosphoprotein associated with GEMs) which binds Csk which is involved in PKA type I signalling as mentioned previously.
  • EBP50 EM-binding phosphoprotein of 50kDa
  • Cbp/PAG Csk-binding protein or phosphoprotein associated with GEMs
  • Ezrin is a 78kDa protein belonging to the ezrin- radixin-moesin (ERM) -family of proteins that plays structural and regulatory roles in the assembly and stabilization of the plasma membrane by linking microfilaments to the membrane (cortical actin layer) .
  • Ezrin is often located at actin-enriched foci; for instance during membrane polarization, lymphocyte migration and polarized secretion of cytokines .
  • ERM proteins have a high degree of homology in their N- terminal regions and bind directly to a number of transmembrane proteins including CD44, layilin, Na/H exchanger NHE1, CD43 and intercellular adhesion molecules (ICAMs) (Tsukita S, 1994, J.
  • IAMs intercellular adhesion molecules
  • the N-terminal ERM domain also binds to signalling molecules in the Rho pathway, including Rho guanine dinucleotide dissociation domain (Rho-GDI) (Hirao et al . , 1996, J. Cell. Biol., 135, p37-51) .
  • Rho-GDI Rho guanine dinucleotide dissociation domain
  • the carboxyl terminus of ERM proteins interacts with filamentous actin (F-actin) and thereby links the cytoskeleton to the plasma membrane.
  • ERM proteins Dependent on the phosphorylation status, ERM proteins can undergo intracellular interactions by association of the N- and carboxyl-termini which leads to masking of binding domains essential for intermolecular interactions .
  • the F-actin binding site and sites for interaction with EBP50 and RhoGDI have been shown to be masked in the dormant, inactive monomer.
  • Ezrin had previously been identified as an AKAP for PKA type II. However, there was no suggestion that this molecule could associate with PKA type I or act as an AKAP for that molecule. The presence of ezrin in lipid rafts had not been observed.
  • EBP50 is an abundant cytoplasmic protein containing 2 N-terminal PDZ domains and a C-terminal domain known to bind ERM-proteins .
  • Cbp/PAG is a Csk- associated membrane adapter protein exclusively localized in lipid rafts. Cbp/PAG-Csk complex formation increases Csk activity through a binding and conformational mechanism.
  • Cbp/PAG is phosphorylated in resting, cells and TCR stimulation induces dephosphorylation and dissociation of Csk which activates Src kinases .
  • Cbp/PAG associates via its C-terminal residues (TRL) to one or both of the PDZ domains in EBP50 (Itoh et al .
  • the present invention provides a variety of modes of interrupting PKA type I-mediated signal transduction by affecting the binding between PKA type I and ezrin, between ezrin and EBP50 and/or between EBP50 and Cbp/PAG.
  • Suitable modes of interruption include the use of inhibitors of those interactions, and modification of the wild-type forms to impair normal binding .
  • the present invention provides a method of altering the PKA type I signalling pathway in a cell by administration of an inhibitor (or a molecule encoding an inhibitor) preferably as defined herein, which reduces or inhibits the binding between one or more of the following binding partners : i) ezrin and PKA type I, ii) ezrin and EBP50, and iii) EBP50 and Cbp/PAG.
  • PKA type I signalling pathway refers to a series of signalling events in which PKA type I is activated resulting in increased kinase activity of this enzyme.
  • This signalling pathway is intended to include molecular events from activation of PKA to end effects such as reduced proliferation or IL-2 production, or intermediate effects such as inactivation of Src kinases.
  • altering the activity of the PKA type I signalling pathway is intended to mean the alteration of one or more signalling elements in the pathway (e.g. to affect its enzymatic or other functional properties) which affects downstream signalling events. Alteration of the signalling elements refers to the ability to form interactions with other molecules, e.g. protein-protein interactions.
  • the ultimate effect is to down-regulate downstream events which typify PKA signalling.
  • Alteration of said signalling pathway may be assessed by determining the extent of activation of a molecule involved in said pathway, e.g. phosphorylation of a kinase, e.g. Csk, or examination of levels of molecules whose levels are dependent on the activity of said pathway, e.g. IL-2.
  • a molecule involved in said pathway e.g. phosphorylation of a kinase, e.g. Csk
  • IL-2 e.g. IL-2
  • binding refers to the interaction or association of at least two moieties in a reversible or irreversible reaction, wherein said binding is preferably specific and selective.
  • Specific binding refers to binding which relies on specific features of the molecules involved to achieve binding, ie. does not occur when a non-specific molecule is used (ie. shows significant binding relative to background levels) and is selective insofar as binding occurs between those partners in preference to binding to any of the majority of other molecules which may be present.
  • a "binding partner” refers to a molecule which recognizes and binds specifically (ie. in preference to binding to other molecules) through a binding site to its binding partner. Such binding pairs when bound together form a complex.
  • a “reduced” binding refers to a decrease in binding, e.g. as manifest by reduced affinity for one another and/or an increased concentration of one of the binding pair required to achieve binding. Reduction includes a slight decrease as well as absolute abrogation of specific binding. A total reduction of specific binding is considered to equate to a prevention of binding.
  • “Inhibited” binding refers to adversely affecting (e.g. by competitive interference) the binding of the binding partners by use of an inhibitor molecule which serves to reduce the partners' binding. A reduction in binding or inhibition of binding may be assessed by any appropriate technique which directly or indirectly measures binding between the binding partners . Thus relative affinity may be assessed, or indirect effects reliant on that binding may be assessed.
  • the binding of the 2 binding partners in isolated form may be assessed in the presence of an inhibitor or by using a modified version of one or more of the binding partners which results from the use of an inhibitor, as described hereinafter.
  • tests may be conducted in which the signalling achieved by the PKA type I pathway is examined (see e.g. Figure 9 which examines IL-2 release) or by assessing disrupted localization as evident from displacement of one or more binding partners from biochemical subcellular fractionation such as lipid raft purification or delocalization evident by immunofluorescent staining and epifluorescence microscopy.
  • Inhibitors according to the present invention are intended for use in affecting the PKA type I signalling pathway.
  • inhibitors may affect PKA type II signalling if used in sufficiently high concentrations .
  • preferred inhibitors are those which affect only the PKA type I signalling pathway and not the PKA type II signalling pathway regardless of the concentration used.
  • Inhibitors which may be used are preferably selective for PKA type I relative to PKA type II signalling. If said inhibitors are able to reduce or inhibit both PKA type I and PKA type II signalling, they may be used at an effective concentration such that they are selective for the PKA type I pathway relative to the PKA type II pathway. Appropriate concentrations may be determined in the above described or other appropriate tests. Especially preferably the inhibitors are selective for (or used at concentrations which are selective for) PKA type Io. relative to PKA type I ⁇ signalling.
  • selectivity is present (for inhibition of e.g. type I relative to type II or type Io. relative to type I ⁇ ) when at least a 5-fold lower concentration of said inhibitor is required to reduce binding between the binding partners by 50%. Especially preferably at least a 10 or 100 fold lower concentration is required.
  • said binding may be assessed according to the K D between the binding partners .
  • Said binding may alternatively be assessed according to the K D between the inhibitor and the binding site of the relevant binding partner.
  • the K D should be l-200nM when assessed in vitro .
  • the amino acid sequence of the human form of ezrin appears in SEQ ID NO.
  • the amino acid sequence of the human form of EBP50 appears in SEQ ID NO . 3 : msadaaagap Iprlcclekg pngygfhlhg ekgklgqyir Ivepgspaek agllagdrlv evngenveke thqqwsrir aalnavrllv vdpetdeqlq klgvqvreel lraqeapgqa 1 eppaaaevqg agneneprea dkshpeqrel rprlctmkkg psgygfnlhs dkskpgqfir 1 svdpdspaea sglraqdriv evngvcmegk qhgdwsair aggdetkllv vdretdeffk 1 kcrvipsqeh Ingplpvpft
  • the binding partners ezrin, PKAI, EBP50 and Cbp/PAG as described herein refer to a polypeptide comprising SEQ ID NO . 1, 2, 3 and 4, respectively, and their functionally equivalent variants, derivatives or fragments.
  • the binding partners are those molecules which occur endogenously .
  • variants, derivatives and fragments are described hereinafter with particular reference to inhibitors of the invention.
  • variants, derivatives and fragments of the binding partners as mentioned herein are similarly defined.
  • such variants include naturally occurring variants such as comparable proteins found in other species or more particularly variants and alleles found within humans. Conveniently, said variants may be described as having more than 75%, e.g.
  • the present invention provides a method of altering the PKA type I signalling pathway in a cell by administration of an inhibitor (or a molecule encoding an inhibitor) as defined herein, which reduces or inhibits binding between one or more of the following binding partners : i) a polypeptide comprising the sequence as set forth in SEQ ID No. 1 or a sequence with 95% similarity thereto or a sequence encoded by a nucleotide sequence which hybridizes under conditions of high stringency to the nucleotide sequence encoding the amino acid sequence of SEQ ID No.
  • polypeptide comprising the sequence as set forth in SEQ ID No. 2 or a sequence with 95% similarity thereto or a sequence encoded by a nucleotide sequence which hybridizes under conditions of high stringency to the nucleotide sequence encoding the amino acid sequence of SEQ ID No . 2 , or a functionally equivalent fragment thereof; ii) a polypeptide comprising the sequence as set forth in SEQ ID No. 1 or a sequence with 95% similarity thereto or a sequence encoded by a nucleotide sequence which hybridizes under conditions of high stringency to the nucleotide sequence encoding the amino acid sequence of SEQ ID No .
  • polypeptide comprising the sequence as set forth in SEQ ID No. 3 or a sequence with 95% similarity thereto or a sequence encoded by a nucleotide sequence which hybridises under conditions of high stringency to the nucleotide sequence encoding the amino acid sequence of SEQ ID No . 3 , or a functionally equivalent fragment thereof; iii) a polypeptide comprising the sequence as set forth in SEQ ID No. 3 or a sequence with 95% similarity thereto or a sequence encoded by a nucleotide sequence which hybridizes under conditions of high stringency to the nucleotide sequence encoding the amino acid sequence of SEQ ID No.
  • sequence similarity refers to sequences which have the stated value when assessed using e.g.
  • sequences encoded by a sequence which hybridizes to a particular sequence refers to the polypeptide sequence encoded by the complementary sequence of a sequence which hybridizes to the particular sequence.
  • hydridizing sequences may be described as those which exhibit at least 70%, preferably at least 80 or 90%, e.g. at least 95% sequence identity (as determined by, e.g.
  • binding partners consist of, or comprise, specific fragments (functionally equivalent fragments) of said above described polypeptides which correspond to the relevant binding sites .
  • Ezrin binds to PKA type I through bases in the region of 356 to 470 (particularly in the region 363 to 470) .
  • residues 413 or 414 to 443 (containing the amphipathic helix binding domain at residues 413-430) are involved in binding to PKA type I.
  • a second region has also been identified that provides specificity and additional affinity in anchoring the enzyme. This region is found at residues 356 to 397 (particularly 367 to 390) and is described in more detail in Example 3 and Figure 10. This region has been found to comprise two separate binding elements, namely from residues 356 to 376 and 378 to 397 (see Example 4) . Binding to PKA involves at least residues 1-50 of that enzyme. Residues 1-286 of ezrin bind to residues 329-358 of EBP50.
  • said binding partner polypeptide consists of at least the following amino acid sequence or a sequence with 95% similarity thereto or a sequence encoded by a nucleotide sequence which hybridizes under conditions of high stringency thereto: (a) amino acids 356 to 470, (e.g. 363-470) (for binding to SEQ ID No. 2 or its variants) or 1 to 286 (for binding to SEQ ID No . 3 or its variants) of SEQ ID No. 1; (b) amino acids 1 to 50 of SEQ ID No . 2 (for binding to SEQ ID No.
  • binding may be reduced by modifying the endogenous molecules taking part in binding.
  • Both inhibitors and the molecules used to achieve an alteration in the form or expression of one or more of the endogenous binding partners are referred to herein as inhibitors even though in the latter case their mode of interaction is not necessarily to achieve direct inhibition of the binding site (ie. a steric inhibitor) .
  • molecules which act to ultimately achieve inhibition of binding between binding partners are referred to herein as inhibitors .
  • Such molecules include peptides and proteins as well as nucleic acid molecules, such as those which encode peptide/protein inhibitors which may be used to express the peptide/protein inhibitors within the cell or may be used to derive sense of antisense nucleotide sequences to cause co-suppression or suppression to reduce expression of the endogenous binding partner.
  • the present invention provides an inhibitor molecule which reduces or inhibits the binding between one or more of the binding partners as defined hereinbefore and which preferably is capable of altering the PKA type I signalling pathway in a cell.
  • direct inhibitors of binding are antagonists of binding between the specific binding pairs .
  • Such inhibitors may themselves bind to one of the binding pair at the binding site (e.g.
  • an "antagonist” is a molecule or complex of molecules which by virtue of structural similarity to one molecule of a binding pair competes with that molecule for binding to the other molecule of the binding pair.
  • the inhibitors may be molecules which specifically recognize the binding site, such as antibodies (or fragments thereof) , or proteins or peptides which associate with that region or associate sufficiently close to affect accessibility by the other binding partner.
  • molecules, particularly peptides or larger molecules, which mimic the binding site (or contain a region which mimics the binding site) may be used to act instead as the pseudo-binding partner to reduce the extent of binding between the true endogenous binding partners .
  • Preferred mimics are peptides that comprise the relevant binding site as described herein.
  • Molecules which are complementary to the binding site, e.g. such that they bind to that site may also be used.
  • Molecules which affect the binding site indirectly, by binding at a distant part of the molecule, but which affect the conformation of the binding site and thus the ability to form relevant interactions with its binding partner may also be used.
  • Appropriate molecules may for example be proteins or peptides or other molecules which can affect binding, or a nucleic acid molecule which encodes such a product may be used to generate the inhibitor. Conveniently small inhibitor molecules may be used. However, where desired, large molecules containing the binding site may be employed, e.g. larger molecules which mimic the entire binding partner (e.g. the soluble portion thereof) , but which have preferably been modified such that one or more properties required for involvement in the signalling pathway is missing or altered to lose or impair its ability to be involved in signalling. For example in the case of EBP50 a large molecule absent only the 30 C-terminal amino acids may be used, which would allow functional binding with Cbp/PAG, but not ezrin.
  • the present invention further extends to an inhibitor which is a polypeptide (or the nucleic acid molecules encoding it) containing one or more mutations in one or more binding sites described herein, in which said mutation results in a molecule which has impaired binding at that site to the relevant binding partner relative to the same molecule without the mutation.
  • one or more of the valine and/or isoleucine residues in the PKA binding site of the ezrin molecule may be mutated, preferably to a proline residue, and the resultant molecule used as an inhibitor to interfere with the ability of the endogenous enzyme to bind to its binding partner PKA.
  • the present invention thus also extends to novel modified, e.g. mutated binding partners or functionally equivalent variants, derivatives or fragments thereof and the nucleic acid molecules which encode them.
  • the present invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding a binding partner, or a functionally equivalent variant, derivative or fragment thereof, as defined above, wherein said sequence is modified as defined above to alter its ability to bind to its binding partner as defined above.
  • Appropriate inhibitors for use in methods of the invention may be identified or tested using appropriate screening tests.
  • the present invention provides a method of screening for, or testing the ability or efficacy of, a molecule to reduce or inhibit binding between any one of the aforementioned binding partners, wherein said test molecule is contacted with said binding partners and the extent of binding is assessed.
  • binding partners are present in isolated form, for example the first of said binding pair may be immobilized on a solid support and the ability of the second of said pair to bind to said first binding partner may be assessed in the presence or absence of said test molecule, ie. by competition.
  • said binding partners may be present in endogenous form, e.g. in a cell and the ability of said test molecule to affect PKA type I signalling by examination of an indicator of said signalling, e.g. T-cell proliferation or IL-2 release, may be examined.
  • Preferred inhibitors of binding between the ezrin and PKA type I binding partners as described hereinbefore mimic the PKA type I binding site on ezrin, ie.
  • inhibitors are derived from and/or mimic said binding site.
  • said inhibitor may comprise or may encode one or more of the residues mentioned above and/or e.g.
  • inhibitors as described herein consist of less than 100 amino acids, e.g. are or encode peptides which consist of less than 100 amino acids, but are preferably shorter, e.g. less than 50, e.g.
  • “Functionally equivalent” variants, derivatives or fragments thereof refers to molecules, preferably peptides, related to, or derived from the above described amino acid sequences, where the amino acid sequence has been modified by single or multiple amino acid (e.g. at 1 to 10, e.g. l to 5, preferably 1 or 2 residues) substitution, addition and/or deletion or chemical modification, including deglycosylation or glycosylation, but which nonetheless retain functional activity, insofar as they act as inhibitors, e.g.
  • substitution variants are included amino and/or carboxyl terminal fusion proteins or polypeptides, comprising an additional protein or polypeptide or other molecule fused to the inhibitor (or binding partner) sequence.
  • substitution variants preferably involve the replacement of one or more amino acids with the same number of amino acids and making conservative substitutions .
  • Such functionally-equivalent variants mentioned above include in particular naturally occurring biological variations (e.g. allelic variants or geographical variations within a species, most particularly variants and alleles found within humans) and derivatives prepared using known techniques.
  • inhibitors (or binding partners) described herein extend to inhibitors (or binding partners) which are functional in (or present in) , or derived from proteins isolatable from, different genera or species than the specific inhibitors and binding partner molecules mentioned herein.
  • Preferred "derivatives" or “variants” include those in which instead of the naturally occurring amino acid the amino acid which appears in the sequence is a structural analog thereof .
  • Amino acids used in the sequences may also be derivatized or modified, e.g. labelled, glycosylated or methylated, providing the function of the inhibitor (or binding partner) is not significantly adversely affected.
  • Derivatives particularly include peptidomimetics which may be prepared using techniques known in the art.
  • non-standard amino acids such as ot- aminobutyric acid, penicillamine, pyroglutamic acid or conformationally restricted analogs, e.g. such as Tic (to replace Phe) , Aib (to replace Ala) or pipecolic acid (to replace Pro) may be used.
  • Tic to replace Phe
  • Aib to replace Ala
  • pipecolic acid to replace Pro
  • Other alterations may be made when the inhibitor is to be used in the methods of the invention described hereinafter (or the binding partners are to be used for screening for inhibitors) . In such cases, the stability of the inhibitor (or binding partner), e.g. peptide, may be enhanced, e.g.
  • D-amino acids or amide isosteres (such as N- methyl amide, retro-inverse amid, thioamide, thioester, phosphonate, ketomethylene, hydroxymethylene, fluorovinyl, (E) -vinyl, methyleneamino, methylenethio or alkane) which protect the peptides against proteolytic degradation.
  • Di (oligo) peptidomimetics may also be prepared.
  • Precursors of the inhibitors (or binding partners) are also encompassed by the term functionally equivalent variants and include molecules which are larger than the inhibitors (or binding partners) and which may optionally be processed, e.g. by proteolysis to yield the inhibitor (or binding partner) .
  • Additional moieties may also be added to the inhibitors (or binding partners) to provide a required function, e.g. a moiety may be attached to assist or facilitate entry of the inhibitor into the cell .
  • Derivatives and variants as described above may be prepared during synthesis of the inhibitor (or binding partner if isolated binding partners are to be used) , e.g. peptide, or by post-production modification, or when the peptide is in recombinant form, using the known techniques of site-directed mutagenesis including deletion, random mutagenesis and/or ligation of nucleic acids .
  • Functionally-equivalent "fragments" according to the invention may be made by truncation, e.g.
  • fragments may be derived from the inhibitor peptides (or binding partners) described above, or may be derived from a functionally equivalent peptide as described above, but which retain the ability to act as an inhibitor (or binding partner) according to the method of the invention.
  • fragments are between 6 and 30 residues in length, e.g. 6 to 25 or 10 to 15 residues.
  • these fragments satisfy the homology (relative to a comparable region) or hybridizing conditions mentioned herein.
  • functional variants according to the invention have an amino acid sequence which has more than 75%, e.g. 75 or 80%, preferably more than 85%, e.g.
  • nucleotide sequences are the degenerate sequences which encode the recited polypeptides or their variants or fragments .
  • peptide inhibitors in this aspect comprise the following sequence or a functionally equivalent variant, derivative or fragment thereof, based on residues 413- 430:
  • each of Xi to X s is any amino acid, wherein Xi is preferably arginine, glutamic acid, glutamine, threonine, phenylalanine, lysine or tyrosine, especially preferably glutamine;
  • X 2 is preferably arginine, lysine, glycine, phenylalanine, alanine, serine or asparagine, especially preferably alanine;
  • X 3 is preferably arginine, glutamic acid, glutamine, threonine, lysine, serine, valine, alanine or leucine, especially preferably glutamic acid;
  • X 4 is preferably glutamic acid, glutamine, lysine, leucine, serine, aspartic acid, alanine, glycine or asparagine, especially preferably glutamic acid;
  • X 5 is preferably tyrosine;
  • X s is preferably thre
  • said sequence is:
  • said sequence may be based on ezrin residues 367-385 and may have the form: D/EX 1 X 2 X 3 X 4 X 5 LX 6 X 7 X 8 EEX 9 X ;L0 X 11 X. L2 X 13 EE
  • each of X_ and X 2 is any amino acid, wherein X ⁇ is preferably arginine, glutamine, alanine, glutamic acid or isoleucine, preferably glutamine; X 2 is preferably asparagine, isoleucine, glutamic acid or glutamine, preferably isoleucine; and wherein X 3 is glutamic acid, glutamine, valine, arginine or phenylalanine, preferably glutamine; X 4 is glutamic acid, arginine, leucine or methionine, preferably arginine; X s is glycine, alanine, arginine or glutamic acid, preferably alanine,- X 6 is aspartic acid, glutamine, lysine or arginine, preferably glutamine; X 7 is arginine, leucine, methionine or lysine, preferably leucine; X B is asparagine, glutamic acid,
  • said sequence is : D/ EX I X 2 X 3 X 4 ALQX 7 EEEX X ⁇ QRX I X I3 EE .
  • inhibitors or their encoding nucleic acid sequences form further preferred aspects of the invention for use in methods of the invention such as a method of altering the PKA type I signalling pathway in a cell by administration of said inhibitor.
  • Preferred inhibitors of binding between the ezrin and EBP50 binding partners as described hereinbefore mimic the ezrin binding site on EBP50 or the EBP50 binding site on ezrin, ie. residues 329 to 358 of SEQ ID No. 3 (EBP50) , or residues 1-286 of SEQ ID No. 1 (ezrin) , respectively, or variants as described hereinbefore .
  • the inhibitors are derived from and/or mimic said binding sites.
  • said inhibitor may comprise or encode one or more of residues 1-20, 20- 40, 40-60, 60-80, 80-100, 100-120, 120-140, 140-160, 160-180, 180-200, 200-220, 220-240, 240-260 or 260-286 of SEQ ID No. 1 or 329 to 349 or 339 to 358 of SEQ ID No. 3 or functionally equivalent variants, derivatives or fragments of such peptides as described hereinbefore.
  • Preferred inhibitors of binding between the EBP50 and Cbp/PAG binding partners as described hereinbefore mimic the EBP50 binding site on Cbp/PAG or the Cbp/PAG binding site on EBP50, ie . 429 to 432 residues of SEQ ID No.
  • inhibitors are derived from and/or mimic said binding sites.
  • said inhibitor may comprise or encode one or more of residues 429-432 of SEQ ID No. 4 or residues 11-30, 31-50, 51-70, 71-90, 80-97, 149-170, 171-190, 191-210, 211-230 or 215-236 of SEQ ID No. 3 or functionally equivalent variants, derivatives or fragments of such peptides as described hereinbefore .
  • inhibitors may where appropriate include full length molecules identical or closely related to those found endogenously to compete with the wild-type molecule.
  • appropriate inhibitors include any of the aforementioned binding partner proteins in complete form.
  • the present invention also extends to antibodies (monoclonal or polyclonal) and their antigen-binding fragments (e.g F(ab) 2 , Fab and Fv fragments ie. fragments of the "variable" region of the antibody, which comprises the antigen binding site) directed to the binding sites or inhibitor peptides as defined hereinbefore, ie .
  • inhibitors as described herein are conveniently proteinaceous, however the use of other types of inhibitors of the binding is contemplated, e.g. molecules which match the binding sites' spatial and conformational structure.
  • inhibitor molecules as described hereinbefore are conveniently added to a cell . This may be achieved by relying on spontaneous uptake of the inhibitor into the cells or appropriate carrier means may be provided.
  • Exogenous peptides or proteins may thus be introduced by any suitable technique known in the art such as in a liposome, niosome or nanoparticle or attached to a carrier or targetting molecule (see hereinafter) .
  • the protein may be tagged with a suitable sequence that allows the protein to cross the cell membrane.
  • An example of such a tag is the HIV tat sequence or a stretch of e.g. 11 arginines . It will be appreciated that the level of exogenous molecules introduced into a cell will need to be controlled to avoid adverse effects .
  • the inhibitor may be transported into the cell in the form of the inhibitor or in the form of a precursor, e.g.
  • the inhibitors may be administered to a cell by transfection of a cell with a nucleic acid molecule encoding the peptide or protein inhibitor.
  • the present invention thus extends to nucleic acid molecules comprising a sequence which encodes the peptide/polypeptide inhibitors described herein and their use in methods described herein.
  • said nucleic acid molecules are contained in a vector, e.g. an expression vector.
  • the nucleic acid molecules described above may be operatively linked to an expression control sequence, or a recombinant DNA cloning vehicle or vector containing such a recombinant DNA molecule.
  • This allows intracellular expression of the inhibitor as a gene product, the expression of which is directed by the gene(s) introduced into cells of interest.
  • Gene expression is directed from a promoter active in the cells of interest and may be inserted in any form of linear or circular DNA vector for incorporation in the genome or for independent replication or transient transfection/expression. Alternatively, the naked DNA molecule may be injected directly into the cell.
  • Appropriate expression vectors include appropriate control sequences such as for example translational (e.g.
  • vectors may include plasmids and viruses (including both bacteriophage and eukaryotic viruses) .
  • Suitable viral vectors include baculovirus and also adenovirus, adeno-associated virus, herpes and vaccinia/pox viruses . Many other viral vectors are described in the art.
  • Preferred vectors include bacterial and mammalian expression vectors pGEX-KG, pEF- neo and pEF-HA.
  • the nucleic acid molecule may conveniently be fused with DNA encoding an additional polypeptide, e.g. glutathione-S-transferase, to produce a fusion protein on expression.
  • an additional polypeptide e.g. glutathione-S-transferase
  • the present invention provides a vector comprising a nucleic acid molecule as defined above.
  • Other aspects of the invention include methods for preparing recombinant nucleic acid molecules according to the invention, comprising inserting nucleotide sequences encoding the modified substrate into vector nucleic acid.
  • Nucleic acid molecules of the invention preferably contained in a vector, may be introduced into a cell by any appropriate means . Suitable transformation or transfection techniques are well described in the literature.
  • a variety of techniques are known and may be used to introduce such vectors into prokaryotic or eukaryotic cells for expression.
  • Preferred host cells for this purpose include insect cell lines, eukaryotic cell lines or E. coli , such as strain BL21/DE3.
  • the invention also extends to transformed or transfected prokaryotic or eukaryotic host cells containing a nucleic acid molecule, particularly a vector as defined above.
  • a further aspect of the invention provides a method of preparing a peptide or protein inhibitor of the invention as hereinbefore defined, which comprises culturing a host cell containing a nucleic acid molecule as defined above, under conditions whereby said inhibitor is expressed and recovering said inhibitor thus produced.
  • the expressed protein product forms a further aspect of the invention.
  • the invention also extends to an inhibitor encoded by a nucleic acid molecule as hereinbefore described. This may be produced by expression of a host cell as described above. Cells containing inhibitors of the invention, introduced directly as inhibitors or by expression of encoding nucleic acid material form further aspects of the invention. Nucleic acid molecules which may be used according to the invention may be single or double stranded DNA, cDNA or RNA, preferably DNA and include degenerate, substantially homologous and hybridizing sequences as described before. Ideally however genomic DNA or cDNA is employed. Inhibitors as described above may be prepared by conventional modes of synthesis including genetic or chemical means .
  • Chemical syntheses may be performed by methods well known in the art involving in the case of peptides cyclic sets of reactions of selection deprotection of the functional groups of a terminal amino acid and coupling of selectively protected amino acid residues, followed finally by complete deprotection of all functional groups. Synthesis may be performed in solution or on a solid support using suitable solid phases known in the art, such as the well known Merrifield solid phase synthesis procedure.
  • the inhibitors of the invention are substantially purified, e.g. pyrogen-free, e.g. more than 70%, especially preferably more than 90% pure (as assessed for example, in the case of peptides or proteins, by an appropriate technique such as peptide mapping, sequencing or chromatography) .
  • Purification may be performed for example by chromatography (e.g. HPLC, size-exclusion, ion-exchange, affinity, hydrophobic interaction, reverse-phase) or capillary electrophoresis .
  • Alternative methods of reducing binding between the binding partners as defined hereinbefore includes modification of endogenous molecules taking part in said binding.
  • the invention extends to modifying the endogenous binding partner as described hereinbefore in a cell . This may be achieved for example by manipulation of the wild-type gene, by manipulating expression of the gene (e.g. by affecting transcription or translation) or by manipulating the expressed product. This could for example be achieved by using antisense oligonucleotides comprising nucleic acid sequences as described hereinbefore (ie.
  • binding partners or of relevant protein/peptide inhibitors or their complementary sequences, ribozymes, RNAi or antibodies and the invention extends to such molecules and their uses.
  • this could be performed for example by somatic cell gene therapy with homologous recombination to for example remove or mutate the binding site.
  • This could be performed on for example hematopoietic stem cells or on blood cells ex vivo or in vivo .
  • Mutation of one or more of the leucine or valine residues to a proline residue in the ezrin binding site for PKA for example could be performed to generated proteins that have reduced binding to PKA.
  • wild-type or mutated sequences may be used to cause co-suppression of the naturally occurring molecule.
  • exogenous molecules may be administered to cells as described hereinbefore.
  • the above described methods of the invention may be used in vi tro, for example in cell or organ culture, particularly for affecting PKA type I signalling pathways which have been activated or to reduce the extent of endogenous signalling.
  • the method may also be used ex vivo, on animal parts or products, for example organs or collected blood, cells or tissues, particularly when it is contemplated that these will be reintroduced into the body from which they are derived.
  • levels may be normalized, e.g.
  • sample refers to any material obtained from a human or non-human animal, including tissues and body fluid.
  • Body fluids in this case include in particular blood, spinal fluid and lymph and "tissues” include tissue obtained by surgery or other means .
  • tissues include tissue obtained by surgery or other means .
  • Such methods are particularly useful when the inhibitor is to be introduced into the body by expression of an appropriate nucleic acid molecule, or the inhibitor is itself a nucleic acid molecule. T cells for example, could be treated in this way.
  • the methods of treatment of the invention as described hereinafter comprise the initial step of obtaining a sample from an individual, contacting cells from said sample with an inhibitor (or a nucleic acid molecule encoding an inhibitor) of the invention and administering said cells of said sample to the individual .
  • the step of contacting refers to the use of any suitable technique which results in the presence of said inhibitor in cells of the sample.
  • the method may also be used in vivo for the treatment or prevention of diseases in which abnormal PKA type I signalling occurs and this will be discussed in more detail below. As described previously the methods of altering PKA type I signalling have utility in a variety of clinical indications in which abnormal PKA type I signalling is exhibited.
  • the inhibitors which abolish the function of PKA type I may be used to produce pharmaceutical preparations to treat immunosuppressive disease.
  • the inhibitors may be used to treat or prevent disorders typified by aberrant PKA type I signalling such as immunosuppressive disorders (such as HIV infection, AIDS or common variable immunodeficiency) or proliferative diseases in which PKA type I signalling has been implicated, e.g. cancers such as colorectal carcinoma, pancreatic carcinoma, hepatocellular carcinoma, cancer mamma, ovarian cancer and non-small cell carcinoma of the lung) .
  • the inhibitors as described herein may therefore be formulated as pharmaceutical compositions in which the inhibitor may be provided as a pharmaceutically acceptable salt.
  • Pharmaceutically acceptable salts may be readily prepared using counterions and techniques well known in the art.
  • the invention thus further extends to pharmaceutical compositions comprising one or more inhibitors (e.g. nucleic acid molecules, peptides or proteins, such as an antisense oligonucleotide, ribozyme or antibody, nucleic acid molecule or peptide/protein) as defined above and one or more pharmaceutically acceptable excipients and/or diluents .
  • pharmaceutically acceptable is meant that the ingredient must be compatible with other ingredients in the composition as well as physiologically acceptable to the recipient.
  • the active ingredient for administration may be appropriately modified for use in a pharmaceutical composition.
  • peptides such as peptidomimetics as described hereinbefore.
  • the active ingredient may also be stabilized for example by the use of appropriate additives such as salts or non-electrolytes, acetate, SDS, EDTA, citrate or acetate buffers, mannitol, glycine, HSA or polysorbate.
  • Conjugates may be formulated to provide improved lipophilicity, increase cellular transport, increase solubility or allow targeting. Conjugates may be made terminally or on side portions of the molecules, e.g. on side chains of amino acids.
  • conjugates may be cleavable such that the conjugate behaves as a pro-drug. Stability may also be conferred by use of appropriate metal complexes, e.g. with Zn, Ca or Fe .
  • the active ingredient may be formulated in an appropriate vehicle for delivery or for targeting particular cells, organs or tissues.
  • the pharmaceutical compositions may take the form of microemulsions, liposomes, niosomes or nanoparticles with which the active ingredient may be absorbed, adsorbed, incorporated or bound. This can effectively convert the product to an insoluble form.
  • These particulate forms have utility for transfer of nucleic acid molecules and/or protein/peptides and may overcome both stability (e.g. enzymatic degradation) and delivery problems .
  • These particles may carry appropriate surface molecules to improve circulation time (e.g. serum components, surfactants, polyoxamine908 , PEG etc.) or moieties for site-specific targeting, such as ligands to particular cell borne receptors .
  • Appropriate techniques for drug delivery and for targeting are well known in the art and are described in W099/62315.
  • site directed targeting see for example Schafer et al . , 1992, Pharm. Res., 9, p541-546 in which nanoparticles can be accumulated in HIV-infected macrophages.
  • Such derivatized or conjugated active ingredients are intended to fall within the definition of inhibitory molecules which form aspects of this invention.
  • compositions for use according to the invention may be formulated in conventional manner using readily available ingredients.
  • the active ingredient may be incorporated, optionally together with other active substances as a combined preparation, with one or more conventional carriers, diluents and/or excipients, to produce conventional galenic preparations such as tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions (as injection or infusion fluids) , emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium) , ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions, sterile packaged powders, and the like.
  • Biodegradable polymers may also be used for solid implants.
  • the compositions may be stabilized by use of freeze-drying, undercooling or Permazyme .
  • Suitable excipients, carriers or diluents are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, calcium carbonate, calcium lactose, corn starch, aglinates, tragacanth, gelatin, calcium silicate, macrocrystalline cellulose, polyvinylpyrrolidone, cellulose, water syrup, water, water/ethanol, water/glycol, water/polyethylene, glycol, propylene glycol, methyl cellulose, methylhydroxybenzoates, propyl hydroxybenzoates, talc, magnesium stearate, mineral oil or fatty substances such as hard fat or suitable mixtures thereof.
  • compositions may additionally include lubricating agents, wetting agents, viscosity increasing agents, colouring agents, granulating agents, disintegrating agents, binding agents, osmotic active agents, emulsifying agents, suspending agents, preserving agents, sweetening agents, flavouring agents, adsorption enhancers, e.g. for nasal delivery (bile salts, lecithins, surfactants, fatty acids, chelators) and the like.
  • lubricating agents wetting agents, viscosity increasing agents, colouring agents, granulating agents, disintegrating agents, binding agents, osmotic active agents, emulsifying agents, suspending agents, preserving agents, sweetening agents, flavouring agents, adsorption enhancers, e.g. for nasal delivery (bile salts, lecithins, surfactants, fatty acids, chelators) and the like.
  • compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration of the patient by employing procedures well known in the art .
  • the active ingredient in such compositions may comprise from about 0.01% to about 99% by weight of the formulation, preferably from about 0.1 to about 50%, for example 10%.
  • the invention also extends to pharmaceutical compositions as described above for use as a medicament. In methods of the invention, inhibitors should be used at appropriate concentrations such that a significant number of the relevant binding partners' interactions, are prevented.
  • the pharmaceutical composition is formulated in a unit dosage form, e.g. with each dosage containing from about 0.1 to 500mg of the active ingredient.
  • an effective dose may lie in the range of from about O.Olmg/kg to 20mg/kg, depending on the animal to be treated, and the substance being administered (e.g 0.1 to 7mg/kg for antisense oligonucleotides) , taken as a single dose.
  • suitable dosages of said inhibitor are 25-100nM or lower, such as 10-50nM or 5-25nM.
  • the administration may be by any suitable method known in the medicinal arts, including for example oral, parenteral (e.g. intramuscular, subcutaneous, intraperitoneal or intravenous) percutaneous, buccal, rectal or topical administration or administration by inhalation.
  • parenteral e.g. intramuscular, subcutaneous, intraperitoneal or intravenous
  • the preferred administration forms will be administered orally, rectally or by injection or infusion.
  • oral administration has its limitations if the active ingredient is digestible. To overcome such problems, ingredients may be stabilized as mentioned previously and see also the review by Bernkop-Schn ⁇ rch, 1998, J. Controlled Release, 52, pl-16. It will be appreciated that since the active ingredient for performance of the invention takes a variety of forms, e.g.
  • compositions may be used for treating or preventing conditions in which the PKA type I signalling pathway is abnormal, in particular when the activity of this pathway is elevated.
  • the present invention provides a method of treating or preventing disorders exhibiting abnormal PKA type I signalling activity, preferably immunosuppressive disorders or proliferative diseases, in a human or non-human animal wherein a pharmaceutical composition as described hereinbefore is administered to said animal .
  • the present invention provides the use of a pharmaceutical composition as defined above for the preparation of a medicament for the treatment or prevention of immunosuppressive disorders or proliferative diseases.
  • a "disorder" or “disease” refers to an underlying pathological disturbance in a symptomatic or asymptomatic organism relative to a normal organism, which may result, for example, from infection or an acquired or congenital genetic imperfection.
  • a “condition” refers to a state of the mind or body of an organism which has not occurred through disease, e.g. the presence of a moiety in the body such as a toxin, drug or pollutant.
  • an “immunosuppressive disorder” refers to a disorder which is typified by impaired function of cells involved in normal immune responses, particularly B and T cells, and is also referred to herein as immunodeficiency or immune dysfunction.
  • immunodeficiency disorders are treated.
  • Preferred conditions for treatment according to the invention include infection by retroviruses, particularly HIV (and infection by related viruses in other animals, e.g.
  • the methods described herein may be used to reverse cAMP hyperactivation that produces T cell dysfunction in immunodeficiencies by interrupting cAMP and PKA-mediated signal transduction in T cells, particularly T cell lipid rafts.
  • proliferative diseases refers to those diseases in which aberrant proliferation of cells occurs.
  • said cells are cells involved in the generation or maintenance of immune responses, particularly B or T cells. Such diseases concern decreases in proliferation relative to normal levels .
  • Subjects which may be treated are preferably mammalian, preferably humans and companion or agricultural animals such as dogs, cats, monkeys, horses, sheep, goats, cows, rabbits, rats and mice.
  • treating refers to the reduction, alleviation or elimination, preferably to normal levels, of one or more of the symptoms of said disease, disorder or condition which is being treated, e.g. infectivity or a reduction or alleviation of immune dysfunction, relative to the symptoms prior to treatment.
  • Preventing refers to absolute prevention, ie . absence of detectable infectious agents, e.g. virus and/or maintenance of normal levels with reference to the extent or appearance of a particular symptom (e.g.
  • the method of treatment according to the invention may advantageously be combined with administration of one or more active ingredients which are effective in treating the disorder or disease to be treated.
  • additional active ingredients are cAMP antagonists e.g. a thiosubstituted cAMP analog (such as derivatives of adenosine-3 ', 5 ' -cyclic monophosphorothioate, Rp isomer, such as Rp-8-Br-cAMPS or Rp-8-Cl-cAMPS) , or COX-2 inhibitors as described in WO02/07721, which is incorporated herein by reference.
  • cAMP antagonists e.g. a thiosubstituted cAMP analog (such as derivatives of adenosine-3 ', 5 ' -cyclic monophosphorothioate, Rp isomer, such as Rp-8-Br-cAMPS or Rp-8-Cl-cAMPS)
  • COX-2 inhibitors as described in WO02/07
  • compositions of the invention may additionally contain one or more of such active ingredients .
  • the present invention provides methods and/or compositions which combine one or more inhibitors as described herein with compounds that improve the tolerability of the active ingredient, especially during long term treatment.
  • Typical compounds include antihistamine and proton pump inhibitors .
  • Typical compounds include antihistamine and proton pump inhibitors .
  • Figure 1 shows the identification of AKAPs in T cell lipid rafts in which lysed Jurkat T cells fractionated on sucrose gradients were resolved on SDS-PAGE, blotted to PVDF-membranes, subjected to Rll-overlay assay (A) and probed with the indicated antibodies (B) .
  • Immunodetection of LAT-antibody was used as a marker for lipid rafts. Lanes refer to fraction numbers following sucrose gradient centrifugation of the lysate and fractionation from the top. Mobility of molecular weight markers are indicated;
  • Figure 2 shows deletional mapping of the PKA anchoring domain in ezrin.
  • Schematic view of ezrin fragments expressed and tested for interaction (A, left) . Depicted are the N-terminal (dark shaded) ; oi-helical (light shaded) and C-terminal (black) domains of Ezrin. Fragments were tested for interaction with Rio. and RIIo; in the yeast-2 -hybrid system by growing on selective media and examining ⁇ -gal (A, middle panel) . GST-linked ezrin fragments as indicated were incubated separately with Rio; and RIIo. proteins and precipitated using glutathione-agarose beads (A, right) .
  • Figure 3 shows the determination of association and dissociation rate constants for the binding of Ezrin to the Rio;-subunit .
  • Figure 4 shows the effect of actin disruption by cytochalasin D on distribution of AKAPs in lipid rafts.
  • Lipid rafts were purified from T lymphocytes by sucrose gradient centrifugation following incubation in the absence or presence of cytochalasin D (80 mM) . The same experiment without cytochalasin treatment was performed in parallel. Fractions were resolved on SDS-PAGE, blotted to PVDF-membranes and probed with the indicated antibodies (A, B two different fractions) . Lanes as indicated in Figure 1. Immunoblotting with anti-LAT antibody indicated lipid raft containing fractions. Molecular weight markers are indicated;
  • Figure 5 shows that ezrin interacts with PKA type Io. in T-cell lipid rafts.
  • Lipid raft fractions of T lymphocytes were isolated, pooled and subjected to precipitation with mouse monoclonal antibodies against Rio, RIIo or control IgG (A) . Precipitates were analyzed by 10% SDS-PAGE followed by immunoblotting with mouse monoclonal antibodies against Ezrin (upper panel) , RIo;, RIIoc and PKA-C subunits (lower panels) . Similarly, lipid raft fractions were subjected to immunoprecipitation with anti-ezrin antibodies, precipitated and immunoblotted with the indicated antibodies (B) ;
  • Figure 6 shows that ezrin colocalizes with the RIo.- subunit of PKA in T cell lipid rafts .
  • Jurkat-T cells were immunostained using (A) a mouse monoclonal antibody against RIo; and (B) rabbit polyclonal antibody against the lipid raft specific protein LAT; rabbit polyclonal antibody against ezrin (D,G) and a mouse monoclonal antibody against RIo; (E) or RIIo (H) . Merged images show overlapping subcellular distribution (C,F,I);
  • Figure 7 shows that the RIo;-subunit of PKA is distributed with VSVG-tagged ezrin.
  • Jurkat T-lymphocytes transfected with cDNA constructs coding for VSVG-tagged (full-length) ezrin were stained with mouse monoclonal antibody VSVG (A) and mouse monoclonal IgG 2A antibody against RIo. (B) . Colocalization of RIo; with Ezrin-VSVG appears as signal in the merged image (C) . Control immunocytochemical analysis showed no antibody signal. Scale bar, 5 ⁇ m;
  • Figure 8 shows that ezrin is colocalized with PKA, EBP50 and Csk in T-cell lipid rafts.
  • Lipid raft fractions of T lymphocytes were isolated, pooled and subjected to precipitation with rabbit polyclonal against Csk or control IgG (A) . Precipitates were analyzed by 10% SDS-PAGE followed by immunoblotting with mouse monoclonal antibodies against ezrin (upper panel) or EBP50 (lower panel) . Similarly, lipid raft fractions were subjected to immunoprecipitation with mouse monoclonal anti-EBP50 antibodies, precipitated and immunoblotted with antibodies to Csk or PKA-C (B) .
  • Figure 8C is a schematic of the complex formed during PKA type I signalling;
  • Figure 9 shows the expression of soluble R-binding domains of ezrin and competition of Ht31 to remove anchored RIx from lipid rafts and disrupt cAMP inhibition of T cell function.
  • T lymphocytes were transfected with mammalian expression vectors encoding PKA-binding domains of ezrin, or empty vector and incubated for 16 h. Subsequently, cells were incubated with or without 50 ⁇ M (15 min) cAMP and next stimulated with anti-CD3£ mAb OKT3 and incubated for 20 h. Supernatants were harvested and IL-2 levels were determined by ELISA and the effect of anchoring disruption on cAMP-mediated inhibition of IL-2 production assessed (A) . The release from inhibition by cAMP was also calculated as fold increase in IL-2 production upon disruption of PKA anchoring (B) ;
  • Figure 10 shows the identification of a putative upstream type I regulatory subunit binding domain (RI- AKAP specifier region) in PKA type I binding AKAPs using bioinformatics .
  • the alignment was performed using the Clustal-W program.
  • the names of the AKAPs and residue numbers of the binding domains are indicated on the right. Boxed text shows conserved residues with similar functional properties.
  • the down-stream amphipathic helix binding domain that may confer binding to both PKA types I and II (albeit with different specificities) is shown.
  • the locations of additional residues with structural similarity are marked by stars,-
  • Figure 11 shows analysis of binding of PKA type I and II to various parts of ezrin by peptide array. Scanning arrays of the ezrin polypeptide were synthesized (Autospot, Intavis AG) where each peptide (spot) represents a 20-mer with a 2-residue frame shift from the previous peptide. The immobilized peptides were analyzed for R binding by either RII- 32 P-overlay with wild type RIIo. or by overlay with RI- 32 P RIo.(A98S) or 1-11, 1-15 or 1-24 N-terminal deletions of RIo;(A98S) as indicated above the columns. Binding of 32 P-labeled RI or RII was detected by autoradiography. Regions that appear to bind in the scan analysis are shown;
  • Figure 12 shows lowered sensitivity to cAMP in inhibition of immune responses in mature T cells with Ezrin knockdown.
  • EXAMPLE 1 Identification of Ezrin as an anchoring protein for PKA type I and EBP50
  • Saccharomyces cerevisiae yeast strains were grown at 30°C in standard liquid YPD medium or minimal SD synthetic medium with appropriate supplement amino acids (Clontech Laboratories, Inc.) .
  • Plasmids were retrans ormed into yeast reporter strain HF7c (genotype: MATa, ura3-52, his3-200, lys2-801, ade2-101, trpl-901, leu2-3, 112, gal4-542, gal80-538, LYS : :GAL-HIS3 , URA3 : : (GAL4 17mers) 3 - CYCl-lacZ from Clontech) with plasmid pGBT-PBR to test for histidine prototrophy and ⁇ -galactosidase activity (Clontech, manual) .
  • the cDNA inserts from the positive clones were sequenced.
  • Ezrin constructs were cloned into the EG202 bait vector and RIo; and RIIo; into the JG4-5 prey vector. Mating assays and detection of interactions were performed as described (Gronholm et al . , 1999, J. Cell Sci., 112, p895-904) . Cell Cul ture, Stimulation, and Transfection of Jurkat TAg
  • the human leukemic T cell line Jurkat TAg a derivative of the Jurkat cell line was kept in logarithmic growth in RPMI medium supplemented with 10% fetal calf serum and antibiotics.
  • T-cells (2 x IO 7 ) in 0.4 ml of Opti-MEM were mixed with 20 ⁇ g of Ht31-cDNA construct in electroporation cuvettes with a 0.4 cm electrode gap (Bio-Rad) and subjected to an electric field of 250 V/cm with 960-microfarad capacitance .
  • the cells were expanded in complete medium and harvested after 20 h.
  • Peripheral blood CD3+ T cells were purified by negative selection from buffycoats from normal healthy donors (Ullevaal University Hospital Blood Center, Oslo, Norway) . Briefly, peripheral blood mononuclear cells were isolated by density gradient (Lymphoprep, NycoMed, Oslo, Norway) centrifugation followed by negative selection using monodisperse magnetic beads directly coated with antibodies to CD14 and CD19 and rat anti- mouse IgG beads coated with antibodies to CD56 and a magnet. Magnetic beads were all from Dynal (Oslo, Norway, cat. no. 111.12, 111.04 and 110.11, respectively) whereas anti-CD56 antibody was from Pharmingen (San Diego, CA, cat. no. 31660. d) . All steps were performed at 4°C. Cell suspensions were analyzed by flow cytometry and routinely shown to consist of more than 90% CD3+ cells.
  • T-cells (5xl0 6 ) in 0.1 ml of Nucleofection solution (Amaxa) were mixed with 2 ⁇ g of each cDNA construct and subjected to electroporation in a Nucleofector (Amaxa) following the manufacturer's protocol. The cells were expanded in complete medium and incubated for 16h at 37°C. 4x 10 s T-cells were treated with 50 ⁇ M cAMP for 15 min (37°C) and activated by the addition of 5 g/ml anti-CD3 ⁇ mAb OKT-3. After 16 h incubation at 37°C the level of secreted IL-2 was determined by ELISA.
  • T-cells or Jurkat T-Cells (3x10 s cells obtained from confluent culture flasks) were washed in PBS, resuspended in 1ml of MES containing 80 ⁇ g/ml Cytochalasin D and pelleted after 30 min at 37°C.
  • T- cells were resuspended in 1 ml ice-cold lysis buffer (25 mM MES, pH 6.5, 100 mM NaCl, 5 mM EDTA, 0,7% Triton X-100 with 1 mM sodium orthovanadate, 1 mM PMSF, 10 mM sodium pyrophosphate, and 50 mM sodium fluoride) containing 0.7% Triton X100 and lysed 10 min on ice.
  • 1 ice-cold lysis buffer 25 mM MES, pH 6.5, 100 mM NaCl, 5 mM EDTA, 0,7% Triton X-100 with 1 mM sodium orthovanadate, 1 mM PMSF, 10 mM sodium pyrophosphate, and 50 mM sodium fluoride
  • the lysate was mixed 1:1 with 80% (w/v) sucrose and placed at the bottom of a 5.2 ml polyallomer centrifuge tube (Beckman Instruments), then carefully overlaid with 2.0 ml of 30% (w/v) sucrose in MNE-buffer and finally with 1 ml of 5% (w/v) sucrose in MNE-buffer.
  • Centrifugation was performed at 4°C in a Beckman SW55Ti rotor (20 h, 46000 rpm) . Twelve 0.4 ml fractions were collected gradually from the top of the gradient, proteins were separated by SDS-PAGE and analyzed by immunoblotting.
  • Ezrin fragments fused to GST were produced in Escherichia coli BL21 cells following stimulation with IPTG (3h) .
  • BL21 cells were pelleted from 10 ml of broth, lysed and after removal of insoluble pellet, glutathione beads were added for lh at 4 Q C. The beads were then washed and finally boiled in SDS-PAGE buffer. Proteins were separated on 10% polyacrylamide gel .
  • RI overlay membranes were blocked in "blotto" overnight at 4°C, 100 nM GST-cleaved RIo: was added to "blotto" and incubated with the membrane for 4-6 h at 4°C. Membranes were then washed with PBS and blocked for Western blot analysis against RIo;.
  • RIIo overlay was performed as previously described (Carr & Scott, 1992, Trends Biochem. Sci., 17, p246-249). Briefly, bovine RIIo; was radiolabelled with [ ⁇ - 3Z P] -ATP and unincorporated ATP was removed by gel filtration. Labelled RIIo was then incubated with the membrane overnight and washed extensively to remove non-specific binding. GST-precipitation assay
  • Fragments of ezrin fused to GST were expressed in Escherichia coli BL21 cells, induced using 0.4 mM IPTG and purified on glutathione-sepharose (Amersham Pharmacia Biotech) .
  • 2.5 ⁇ g of purified ezrin fragments and R subunits were incubated at a 1:1 ratio (50 ⁇ M each) in 100 ⁇ l pull-down buffer (300 mM NaCl, 0.1 % Triton X-100, lmM PMSF, lmM EDTA, 5mM benzamidine, 5 mM DTT, lO ⁇ g/ml of antipain, chymostain, leupeptin and pepstatin A) overnight at 4°C.
  • Biol., 327, p609-618) identified 12 clones that interacted with the full-length but not the deleted RIo; bait. Of the 12 clones, 2 were identified as ezrin, 2 were identified as PAP7 , a itochondrial AKAP for PKA type I as earlier reported (Li et al . , 2001, Mol. Endocrinol . , 15, p2211-2228) and the remaining 8 were checked out as false positives.
  • RII overlay screening of a Jurkat T cell Lambda Zap expression library was performed using radio- labelled RIIo; as a probe.
  • this screening identified AKAPs 95, 79, 149 and a fragment of AKAP450 in T cells, which is consistent with the recent report by Carr and co-workers on PKA type II AKAPs in T cells (Schillace et al . , 2002, supra).
  • the screening with RIo; as a bait identified a different set of AKAPs in T cells to those identified by the RII overlay screening.
  • AKAPs that specifically would serve to anchor the pool of PKA type I identified in T cell lipid rafts and that are involved in the PKA type I- Csk inhibitory pathway that regulates TCR signalling
  • sucrose gradient fractionation of Jurkat T cell lysates and performed far-Western experiments (RII overlay) using radio-labelled PKA RIIo; subunit.
  • Fig. 1A shows the result of this experiment, which identified R- binding proteins of approximately 80 and 150 kDa.
  • AKAP149 The approximately 150-kDa band detected in Fig. 1A was further identified as AKAP149 (D-AKAP1) .
  • Ezrin was identified both in a yeast two- hybrid screen and as an R-binding protein purified from lipid rafts;
  • AKAP149 was less abundant in lipid rafts versus non-raft fractions (compare fractions 4 to 5 and 9 to 12) ; and
  • iii) Disruption of PKA-AKAP149 signalling complexes did not appear to affect signalling through the PKA-Csk-Lck regulatory pathway in T cell lipid rafts (not shown) .
  • lipid rafts from T cells in the presence and absence of the actin-depolymerizing drug cytochalasin D.
  • actin cytoskeleton was disrupted, we observed increased amounts of ezrin in lipid rafts (Fig. 4A)..
  • PKA RIo. and some levels of PKA RIIcc were present in the same raft preparations (Fig. 4A) .
  • the ezrin-binding protein EBP50 also co-migrated with ezrin in the density gradient separation and localised to lipid rafts (Fig. 4B) .
  • ezrin and RIo co-localized well with the expressed ezrin (Figs. 7A-C) .
  • overexpression of ezrin resulted in numerous needle-shaped processes at the cell surface (philopodia) as reported elsewhere earlier.
  • RIo also localized to these needle-shaped ezrin containing processes at the cell surface.
  • EBP50 bridges ezrin and Cbp/PAG to yield a scaffold for the PKA-Csk signalling pathway in T cell lipid rafts
  • EXAMPLE 2 Inhibition of PKA Type I signalling by disruption of ezrin binding
  • Ht31 anchoring disruptor containing the R-binding site from AKAP-Lbc is well established and widely used to compete localization of PKA.
  • Ht31 has a higher affinity for PKA type II, it can also be used to disrupt PKA type I interaction with AKAPs (Fig. 3C, Herberg et al . , 2000, J. Biol. Chem., 298, p329-339) .
  • T cells were transfected with a construct directing the expression of the Ht31 fragment (Lester et al . , 1997, PNAS, 94, pl4942-14947) this displaced both PKA types I and II from lipid rafts (not shown) .
  • Peripheral blood T cells were transfected by nucleofection (Amaxa) with constructs directing the expression of a soluble ezrin fragment containing the cx-helical domain mock-transfected.
  • Transfected and control-transfected cells were either left untreated, activated by cross ligation of the TCR/CD3 complex or pretreated with a concentration of cAMP that produced an 80% maximal inhibition of IL-2 production, and subsequently activated and cultured for 16 hours after which IL-2 levels secreted to the supernatant were assessed.
  • Results from these experiments show that T cells expressing soluble fragments of ezrin were less sensitive to cAMP-mediated inhibition of IL-2 production (Fig 9A) .
  • IL-2 production in the presence of cAMP increased 3-fold by introducing soluble fragments of ezrin (Fig. 9B) .
  • EXAMPLE 3 Identification of a putative upstream type I regulatory subunit binding domain (RI-AKAP specifier region) in AKAPs
  • Peptide arrays were synthesized on cellulose paper by using an Autospot Robot ASP222 (ABiMED, Langenfeld, Germany) as described (Frank, R. , 1992, Tetrahedron 48, 123-132) .
  • R-overlays were conducted as described, using 3Z P-labeled recombinant murine RIIo; (Hausken, Z. E. , et al . , 1998 in Protein Targeting Protocols, ed. Clegg, R. A. (Humana, Totowa, NJ) , Vol. 88, pp. 47-64), recombinant bovine RI (A98S) or deletion mutants.
  • radiolabeled RI as a probe, binding to regions 356-375 and 378-397 was detected. This overlapped with the predicted upstream RI-AKAP specifier region in ezrin. Furthermore, this region appeared to have a tandem binding motif. In contrast, no binding to this region was detected with radiolabeled RII or with any of the RI deletion mutants indicating that the RI-AKAP specifier region interacts with the N-terminus of RIo.
  • RII- 32 P overlay demonstrated binding to the most N-terminal part of this region whereas RI- 32 P overlay gave weak binding to the more C-terminal part of the helix. The RI-binding was enhanced when the N-terminus had been removed.
  • EXAMPLE 5 siRNA-mediated knockdown of Ezrin disrupts cAMP inhibition of T cell proliferation
  • siRNA duplexes targeting different positions within human ezrin mRNA were designed and synthesized in-house. Following initial screening of siRNA activity in cell culture, mismatched control siRNAs for the best siRNA were also designed. SiRNAs are named according to the position of the 5 ' nucleotide of the sense strand relative to the reference sequences of the respective target mRNAs and the sequences are:

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Abstract

The invention provides a method of altering the PKA type I signalling pathway in a cell comprising the step of administering an inhibitor, or a molecule encoding an inhibitor, which reduces or inhibits the binding between one or more of the following binding partners: i) ezrin and PKA type I, ii) ezrin and EBP50, and iii) EBP50 and Cbp/PAG. Also provided are inhibitor molecules for use in the method of the invention and pharmaceutical compositions comprising the inhibitors. Methods of treating disorders or diseases in which abnormal PKA type I signalling occurs such as HIV, AIDS,CVI or cancer using such inhibitors are also provided.

Description

Inhibitors
The present invention relates to inhibitors which abolish the function of cAMP dependent protein kinase (PKA) , type I and their use to produce pharmaceutical preparations to treat or prevent immunosuppressive diseases. More specifically, the present invention provides inhibitors of binding between specific binding partners of the PKA type I complex involved in PKA type I signalling. In particular inhibitors of the binding between ezrin and PKA type I, ezrin and EBP50 and EBP50 and Cbp/PAG are provided. The immune system of mammals has evolved different strategies to defend the organism against ,the variety of potentially* infectious agents. The ability to acquire specific and anamnestic responses against intruders relies on the adaptive immune system. The main players in the adaptive immune system are B and T lymphocytes, and the specific recognition of antigen by these cells is mediated by receptors with some degree of structural similarity, yet which are functionally very different. The different receptor specificities are made possible through somatic rearrangement of a limited number of genes and are clonally distributed. The main strategy of this system is to generate a nearly unlimited number of specificities to cover the recognition of almost any foreign antigen. Immunological memory is partly a result of clonal expansion of subsets of T and B cells reacting with a particular antigen, and enables the organism to respond more quickly at the second encounter with the same antigen. Cell proliferation is used as a marker of immune activation. According to the clonal selection theory, exposure to antigen leads to activation of individual B and T cell clones with corresponding receptor specificities. However, the number of cells with affinity for a certain antigen is a small fraction of the total number of cells (approximately 0.001%). It is therefore crucial that the activated cells are capable of proliferation (clonal expansion) in order to generate an adequate immune response. Thus, proliferation is a very important feature characterizing lymphocyte function and allowing immune activation. In in vi tro experiments, it is possible to activate the entire population of isolated T lymphocytes by using antibodies directed against the antigen receptor complex (TCR/CD3) . This will mimic the in vivo situation in which T cells are immunoactivated to clonal expansion through the antigen receptor. It is known that T cell proliferation is inhibited through the cAMP signalling pathway. Cyclic AMP-dependent protein kinase (PKA) is an enzyme present in all cells. Hormones and neurotransmitters binding to specific receptors stimulate the generation of the second messenger 3 ' , 5 ' - cyclic adenosine monophosphate (cAMP) . Cyclic AMP is one of the most common and versatile second messengers. The best characterized and major downstream effector mechanism whereby cAMP exerts its effects involves binding to and activating PKA. PKA is a serine/threonine protein kinase which phosphorylates a number of different proteins within the cell, and thereby regulates their activity. It is known that PKA regulates a vast variety of cellular processes such as metabolism, proliferation, differentiation and regulation of gene transcription. The great diversity of cellular processes mediated by cAMP and PKA strongly suggests that there exists mechanisms that provide the required sensitivity and specificity of the effector pathway to ensure that rapid and precise signalling processes take place. Specificity can be achieved by tissue- and cell-type specific expression of PKA isoforms with different biochemical properties. However, targeting of PKA isoforms by A- kinase anchoring proteins (AKAPs) provides a higher level of specificity to the signalling process by localizing PKA to defined subcellular sites in close proximity to the substrate. Anchoring of PKA by AKAPs may also tune the sensitivity of the signal pathway by recruiting PKA into multiprotein complexes that include phosphodiesterases and protein phosphatases as well as other signal proteins in addition to PKA (Michel and Scott, 2002, Ann. Rev. Pharmacol. Toxicol . , 42, p235- 257) . PKA is made up of four different subunits, a regulatory (R) subunit dimer and two catalytic (C) subunits. Furthermore, two main classes of PKA isozymes, PKA type I and PKA type II (PKAI and PKAII, respectively) have been described. PKAI and PKAII can be distinguished by their R subunits, designated RI and RII. Isoforms of RI and RII are referred to as Rio*, Rlβ, Rllα and Rllβ. Moreover, the C subunits also exist as isoforms referred to as Co., Cβ and Cy. The different subunits may form multiple forms of PKA (isozymes) with potentially more than 18 different forms . Activation occurs upon binding of cAMP to the R subunits followed by the release of the active catalytic subunit. PKA type II is mainly particulate and associated with AKAPs whereas PKA type I is both soluble and particulate although PKA type I anchoring has remained more elusive. However, PKA type I is recruited to the lipid raft fraction of the cell membrane upon T- cell activation, and colocalizes with the TCR-CD3 complex (Skalhegg et al. , 1994, Science, 263, p84-87) . Lipid rafts are specialised membrane domains enriched in certain lipids, cholesterol and proteins. Their primary function is believed to be in signalling transduction. In particular they are considered to be the site of recruitment for various signalling molecules crucial for T cell activation. PKA is a key negative regulator of lymphocyte function. The present inventors and others have shown that cAMP inhibits T lymphocyte proliferation induced through the T cell antigen receptor/CD3 complex (TCR/CD3) . We have shown that T cells express both PKAI and PKAII. However, only the selective activation of PKAI is sufficient to mediate the inhibitory effect of cAMP. In addition, we have demonstrated that PKAI, but not PKAII, redistributes to, colocalizes with and inhibits signalling through antigen receptors on T and B cells and natural killer cells and regulates mitogenic responses in T and B cells and acute cytotoxic responses in NK cells. PKA type I mediates an inhibitory effect on the T cell activation cascade that involves activation of C- terminal Src kinase (Csk) by phosphorylating residue S364 (Vang et al, 2001, J. Exp. Med., 193, p497-507) . Active Csk subsequently phosphorylates the C-terminal inhibitory tyrosine residue of the Src kinase Lck which reduces its activity and thereby acts as a negative regulator of TCR signalling. The processes which are involved are described in more detail in W099/62315, which is incorporated herein by reference. Thus, PKAI serves as a key negative regulator of lymphocyte functions, e.g. mitogenic and cytotoxic responses initiated through antigen receptors. The inventors' work has led to the conclusion that modulation of normal immune responsiveness by activation of PKA type I is a negative feedback mechanism. Dysregulation of this system may lead to immunological overshoot or impaired immune functions . Both primary and secondary immunodeficiencies cause an increased incidence of opportunistic infections and cancer, and are increasing causes of morbidity and mortality in all parts of the world. Human immunodeficiency virus (HIV) causes a chronic infection leading to severe dysfunction of the immune system with markedly increased incidence of a large number of infections and certain forms of malignancies (e.g. lymphoma and Kaposis' sarcoma) . In many communities in the USA, HIV infection is the leading cause of death among "young" adults . In the developing world this problem is even larger. Next to i unoglobulin (Ig) A deficiency, common variable immunodeficiency (CVI) is the most frequent type of primary immunodeficiency. This form of primary hypogammaglobulinaemia is characterized by the onset of immunodeficiency after the first two years of life, by severely decreased serum IgG levels and recurrent bacterial infections, particularly in the respiratory tract . T cell dysfunction is the immunological hallmark of HIV infection. Defective lymphocyte cytokine production and impaired proliferative responses on stimulation are early signs of immunodeficiency in these patients, manifested even before a decline in CD4+ lymphocytes counts is observed. B cell dysfunction with impaired antibody synthesis is the major immunological characteristic of CVI patients. However, the immunological abnormalities in CVI are not restricted to B cells, but often also involve T cell dysfunction, e.g. impaired proliferative responses on stimulation. The B cells in CVI patients are not necessarily intrinsically defective, and impaired T cell "help" may be of importance for the B cell defects in these patients. T cell dysfunction may also be of importance for certain clinical manifestations in these patients not necessarily related to defective antibody production, e.g. increased incidence of granulomata and malignancies. In terms of current therapies, antiretroviral therapy is the main component in the treatment of HIV- infected patients. However, although potent antiretroviral combination therapy may markedly increase the CD4+ and CD8+ lymphocytes counts in HIV-infected patients, impaired T cell function seems to persist, as indicated in the observations made in Example 1, table I and 2B of WO98/48807. Thus, there is a need for immunomodulating agents in addition to antiretroviral therapy in these patients . Immunoglobulin substitution is the main component in the treatment of CVI patients. However, this substitution therapy does not restore the defective T and B cell function. Furthermore, in some clinical complications, e.g. noncaseating granulomata and persistent viral infections, there is a need for therapy which may more directly enhance T cell function. Although impaired T cell function is a well recognized immunological feature of both HIV infection and CVI, the exact molecular mechanism for this T cell impairment was not known. Therapeutic modalities directed against such intracellular defects are expected to be of major importance in the treatment of these patients and may have the potential to restore important immunological defects in HIV-infected patients and in patients with CVI. Hofmann et al (Aids, Vol 7, p659-664, 1993) and W093/19766 have demonstrated that HIV-seropositive individuals without AIDS show a significant increase in intracellular cAMP levels and PKA activity in crude peripheral blood mononuclear cells (PBMC) from HIV- seropositive subjects. Examination of T cells was reported as data not shown and did not reach significance because of larger variability, probably induced by the T cell purification method. Their study further indicated that adenosine analogues such as 2 ' , 5 ' dideoxyadenosine (ddAdo) reduced cellular cAMP levels in PBMC and increased cell proliferation. This effect was, however, concentration dependent such that concentrations in the range of 6ng/ml were effective and higher concentrations were suppressive or did not further inhibit cAMP levels. Similar effects were not demonstrated in T cells sampled from HIV patients. A simple concentration/response relationship was also not demonstrated. Purified T cells were used in their examples but these cells were sampled from healthy blood donors and purified by positive selection that may lead to premature T cell activation. Cho-Chung et al in W093/21929 have shown treatment applied to cancer cells by antagonising cAMP-dependent protein kinase, by using phosphorothioate derivatives of cAMP. Based on previous observations made and published by the present inventors (Skalhegg et al , 1994, Science, 263, p84; Levy et al, 1996, Eur. J. Immunol., 26, pl290- 1296; Torgersen, 1997, J. Biol . Chem. , 272, p5495-5500) it has been established that activation of protein kinase A type I isozymes (and not type II isozymes) is necessary and sufficient to mediate cAMP-dependent inhibition of immune functions such as T and B cell proliferation induced through the antigen receptor or NK cell cytotoxicity mediated by specific NK receptors. Furthermore, protein kinase A type I redistributes to and colocalizes with the antigen receptor complex upon activation and capping of T and B cells (Skalhegg et al , 1994, supra) . This anchoring of protein kinase A type I supports a role for this specific isozyme in modulation of immune responses mediated through receptors on lymphoid cells. Furthermore, discoveries by the inventors and as described in WO98/48809 show that there is an increased activation of protein kinase A type I in T cells from patients with HIV infection or CVI. The inventors further demonstrated that activation of this isozyme of PKA leads to inhibition of immune function that can be reversed by selectively inhibiting the type I and not the type II isozymes of PKA. Based on these findings, the inventors sought compounds aimed at reversing the inappropriate activation of protein kinase A type I in immunodeficiencies (such as HIV, CVI) and thereby restoring T cell function and immune responsiveness. The present inventors therefore set about developing strategies to specifically increase T cell immune function and reverse T cell dysfunction in human immunodeficiency virus infected and common variable immunodeficiency patients by using suitable compounds interfering with the cAMP/PKA pathway in T cells. Furthermore, as the role of PKA type I as an immune modulator is shared among all lymphoid cells (T cells, B cells, NK cells) disruption of the cAMP/PKA pathway is also expected to be relevant to B and NK cell function. Appropriate mechanisms for improving T cell function as developed by the inventors are described in WO98/48809, which is incorporated herein by reference. The strategies relied on disruption of the cAMP-induced inhibition of T cell immune responses by abolishing PKA type I/RIoc signalling. Specifically, a number of specific mechanisms for disrupting the effects mediated by cAMP dependent protein kinase and hence stimulation of immune function were described. These included the use of PKA isozyme-specific cAMP antagonists, gene function knock out strategies, ribozymes, sequence- specific antisense oligonucleotides and the use of anchoring disrupting competitor peptides to displace protein kinase A type I from its anchoring with the antigen receptor complex. WO98/48809 showed that these entities all interfere with signalling through protein kinase A type I and that they could be used separately or in combination in order to target and abolish the inappropriate activation of protein kinase A type I . In W099/62315, the present inventors described the use of various mechanisms to affect phoshorylation of key components of the PKA type I signalling pathways and in so doing alleviate the inhibition of lymphocyte activation. With regard to the use of anchoring disrupting peptides, specific cAMP-mediated effects at defined subcellular loci had been shown to be dependent on anchoring of PKA type II via hydrophobia interactions with an amphipathic helix domain in AKAPs in close proximity to substrates at that subcellular location. Disruption of anchoring by 22-amino acid competition peptides to the interaction domain, introduced by liposome mediated peptide transfer, had been shown to abolish isozyme-specific effects mediated by PKA type II . No similar effects had however been shown for PKA type I . WO98/48809 then describes specific competitor peptides to displace protein kinase A type I from its anchoring with the antigen receptor complex. In doing so, protein kinase A type I function was inhibited by removal of the activated enzyme from substrates in the antigen receptor complex that are relevant for inhibition of immune function in T cells. The search for new and improved modulators of PKA type I signalling and hence of immune function continues. Surprisingly, the inventors have now identified the involvement of a number of molecules not previously known to be involved in signalling via PKA type I which thus constitute attractive targets to mediate cell signalling. Surprisingly, the inventors have now identified a new AKAP which recruits and binds PKA type I and is hence involved in PKA type I signalling. This molecule's involvement in the signalling pathway provides a number of molecules which may be targeted to intervene in the signalling process. Thus, the inventors have identified that the molecule ezrin is an AKAP responsible for anchoring PKA type I to the TCR-CD3 complex during T cell activation and capping. Ezrin also binds to EBP50 (ERM-binding phosphoprotein of 50kDa) which in turn binds to Cbp/PAG (Csk-binding protein or phosphoprotein associated with GEMs) which binds Csk which is involved in PKA type I signalling as mentioned previously. These molecules act as a scaffold that assembles the complexes required for the cAMP- PKA/Csk-pathway in lipid rafts and has been found to be crucial for signalling via the type I PKA enzyme. Ezrin is a 78kDa protein belonging to the ezrin- radixin-moesin (ERM) -family of proteins that plays structural and regulatory roles in the assembly and stabilization of the plasma membrane by linking microfilaments to the membrane (cortical actin layer) . Ezrin is often located at actin-enriched foci; for instance during membrane polarization, lymphocyte migration and polarized secretion of cytokines . ERM proteins have a high degree of homology in their N- terminal regions and bind directly to a number of transmembrane proteins including CD44, layilin, Na/H exchanger NHE1, CD43 and intercellular adhesion molecules (ICAMs) (Tsukita S, 1994, J. Cell Biol . , 126, p391-401; Yonemura S, 1998, J. Cell Biol., 140, p885- 895; Denker SP, 2000, Mol . Cell., 6, pl425-1436; Bono P., 2001, Mol. Biol. Cell., 12, p891-900; Borowsky et al . , 1998, J. Cell Biol., 143, p429-442) as well as indirectly through the apical scaffolding proteins EBP50/sodium-hydrogen exchanger type 3 kinase A regulatory protein (E3KARP) to the tail of other membrane proteins (Reczek et al . , 1997, J. Cell Biol., 139, pl69-179; Yun et al . , 1998, J. Biol. Chem. , 273, p25856-25863) . The N-terminal ERM domain also binds to signalling molecules in the Rho pathway, including Rho guanine dinucleotide dissociation domain (Rho-GDI) (Hirao et al . , 1996, J. Cell. Biol., 135, p37-51) . The carboxyl terminus of ERM proteins interacts with filamentous actin (F-actin) and thereby links the cytoskeleton to the plasma membrane. Dependent on the phosphorylation status, ERM proteins can undergo intracellular interactions by association of the N- and carboxyl-termini which leads to masking of binding domains essential for intermolecular interactions . Thus far, the F-actin binding site and sites for interaction with EBP50 and RhoGDI have been shown to be masked in the dormant, inactive monomer. Ezrin had previously been identified as an AKAP for PKA type II. However, there was no suggestion that this molecule could associate with PKA type I or act as an AKAP for that molecule. The presence of ezrin in lipid rafts had not been observed. EBP50 is an abundant cytoplasmic protein containing 2 N-terminal PDZ domains and a C-terminal domain known to bind ERM-proteins . Cbp/PAG is a Csk- associated membrane adapter protein exclusively localized in lipid rafts. Cbp/PAG-Csk complex formation increases Csk activity through a binding and conformational mechanism. Cbp/PAG is phosphorylated in resting, cells and TCR stimulation induces dephosphorylation and dissociation of Csk which activates Src kinases . Cbp/PAG associates via its C-terminal residues (TRL) to one or both of the PDZ domains in EBP50 (Itoh et al . , 2002, J. Immunol., p541-544; Brdickova et al . , 2001, FEBS Lett., 507, pl33-136) and EBP50 associates with ezrin via the C-terminal 30 a ino acids of EBP50 and the N-terminal portion of ezrin (Reczek & Bretscher, 1998, J. Biol. Chem. , 273, pl8452-18458) . The present invention provides a variety of modes of interrupting PKA type I-mediated signal transduction by affecting the binding between PKA type I and ezrin, between ezrin and EBP50 and/or between EBP50 and Cbp/PAG. Suitable modes of interruption include the use of inhibitors of those interactions, and modification of the wild-type forms to impair normal binding . In a first aspect therefore, the present invention provides a method of altering the PKA type I signalling pathway in a cell by administration of an inhibitor (or a molecule encoding an inhibitor) preferably as defined herein, which reduces or inhibits the binding between one or more of the following binding partners : i) ezrin and PKA type I, ii) ezrin and EBP50, and iii) EBP50 and Cbp/PAG. The "PKA type I signalling pathway" as referred to herein refers to a series of signalling events in which PKA type I is activated resulting in increased kinase activity of this enzyme. This signalling pathway is intended to include molecular events from activation of PKA to end effects such as reduced proliferation or IL-2 production, or intermediate effects such as inactivation of Src kinases. As referred to herein the phrase "altering the activity of the PKA type I signalling pathway" is intended to mean the alteration of one or more signalling elements in the pathway (e.g. to affect its enzymatic or other functional properties) which affects downstream signalling events. Alteration of the signalling elements refers to the ability to form interactions with other molecules, e.g. protein-protein interactions. The ultimate effect is to down-regulate downstream events which typify PKA signalling. Alteration of said signalling pathway may be assessed by determining the extent of activation of a molecule involved in said pathway, e.g. phosphorylation of a kinase, e.g. Csk, or examination of levels of molecules whose levels are dependent on the activity of said pathway, e.g. IL-2. For use in particularly clinical conditions, down-regulation of the PKA signalling pathway, ie. reversal of the effects of cAMP activation, e.g. to reverse lymphocyte dysfunction, is required. As referred to herein "binding" refers to the interaction or association of at least two moieties in a reversible or irreversible reaction, wherein said binding is preferably specific and selective. Specific binding refers to binding which relies on specific features of the molecules involved to achieve binding, ie. does not occur when a non-specific molecule is used (ie. shows significant binding relative to background levels) and is selective insofar as binding occurs between those partners in preference to binding to any of the majority of other molecules which may be present. As referred to herein a "binding partner" refers to a molecule which recognizes and binds specifically (ie. in preference to binding to other molecules) through a binding site to its binding partner. Such binding pairs when bound together form a complex. As referred to herein a "reduced" binding refers to a decrease in binding, e.g. as manifest by reduced affinity for one another and/or an increased concentration of one of the binding pair required to achieve binding. Reduction includes a slight decrease as well as absolute abrogation of specific binding. A total reduction of specific binding is considered to equate to a prevention of binding. "Inhibited" binding refers to adversely affecting (e.g. by competitive interference) the binding of the binding partners by use of an inhibitor molecule which serves to reduce the partners' binding. A reduction in binding or inhibition of binding may be assessed by any appropriate technique which directly or indirectly measures binding between the binding partners . Thus relative affinity may be assessed, or indirect effects reliant on that binding may be assessed. Thus for example, the binding of the 2 binding partners in isolated form may be assessed in the presence of an inhibitor or by using a modified version of one or more of the binding partners which results from the use of an inhibitor, as described hereinafter. Alternatively tests may be conducted in which the signalling achieved by the PKA type I pathway is examined (see e.g. Figure 9 which examines IL-2 release) or by assessing disrupted localization as evident from displacement of one or more binding partners from biochemical subcellular fractionation such as lipid raft purification or delocalization evident by immunofluorescent staining and epifluorescence microscopy. Inhibitors according to the present invention are intended for use in affecting the PKA type I signalling pathway. Some such inhibitors may affect PKA type II signalling if used in sufficiently high concentrations . As a consequence, preferred inhibitors are those which affect only the PKA type I signalling pathway and not the PKA type II signalling pathway regardless of the concentration used. Inhibitors which may be used are preferably selective for PKA type I relative to PKA type II signalling. If said inhibitors are able to reduce or inhibit both PKA type I and PKA type II signalling, they may be used at an effective concentration such that they are selective for the PKA type I pathway relative to the PKA type II pathway. Appropriate concentrations may be determined in the above described or other appropriate tests. Especially preferably the inhibitors are selective for (or used at concentrations which are selective for) PKA type Io. relative to PKA type Iβ signalling. Preferably in in vitro assays, selectivity is present (for inhibition of e.g. type I relative to type II or type Io. relative to type I β) when at least a 5-fold lower concentration of said inhibitor is required to reduce binding between the binding partners by 50%. Especially preferably at least a 10 or 100 fold lower concentration is required. Conveniently said binding may be assessed according to the KD between the binding partners . Said binding may alternatively be assessed according to the KD between the inhibitor and the binding site of the relevant binding partner. Preferably the KD should be l-200nM when assessed in vitro . The amino acid sequence of the human form of ezrin appears in SEQ ID NO. 1: mpkpinvrvt tmdaelefai qpnttgkqlf dqwktiglr evwyfglhyv dnkgfptwlk1 ldkkvsaqev rkenplqfkf rakfypedva eeliqditqk lfflqvkegi Isdeiycppe21 tavllgsyav qakfgdynke vhksgylsse rlipqrvmdq hkltrdqwed riqvwhaehr81 gmlkdna le ylkiaqdlem yginyfeikn kkgtdlwlgv dalglniyek ddkltpkigf41 pwseirnisf ndkkfvikpi dkkapdfvfy aprlrinkri lqlc gnhel ymrrrkpdti 01 evqqmkaqar eekhqkqler qqletekkrr etverekeqm mrekeelmlr Iqdyeektkk 61 aerelseqiq ralqleeerk raqeeaerle adrmaalrak eelerqavdq iksqeqlaae21 laeytakial leearrrked eveewqhrak eaqddlvktk eelhlvmtap ppppppvyep81 vεyhvqeslq degaeptgys aelssegird drneekrite aeknervqrq Ivtlsselsq41 ardenkrthn diihnenmrq grdkyktlrq irqgntkqri defeal
The amino acid sequence of the human form of PKA type I (Ro. subunit) appears in SEQ ID NO . 2:
1 mesgstaase earslrecel yvqkhniqal lkdsivqlct arperptnafl reyferleke1 eakqiqnlqk agtrtdsred eisppppnpv vkgrrrrgai saevyteeda asyvrkvipk21 dyktmaalak aieknvlfsh lddnersdif damfsvsfia getviqqgde gdnfyvidqg81 etdvyvnnew atsvgeggsf gelaliygtp raatvkaktn vkl gidrds yrrilmgstl41 rkrkmyeefl skvsilesld k erltvada lepvqfedgq kivvqgepgd effiilegsa 01 avlqrrsene efvevgrlgp sdyfgeiall mnrpraatw argplkcvkl drprfervlg61 pcsdilkrni qqynsfvsls v
The amino acid sequence of the human form of EBP50 appears in SEQ ID NO . 3 : msadaaagap Iprlcclekg pngygfhlhg ekgklgqyir Ivepgspaek agllagdrlv evngenveke thqqwsrir aalnavrllv vdpetdeqlq klgvqvreel lraqeapgqa 1 eppaaaevqg agneneprea dkshpeqrel rprlctmkkg psgygfnlhs dkskpgqfir 1 svdpdspaea sglraqdriv evngvcmegk qhgdwsair aggdetkllv vdretdeffk 1 kcrvipsqeh Ingplpvpft ngeiqkensr ealaeaales prpalvrsas sdtseelnsq 01 dsppkqdsta psstsssdpi ldfnislama kerahqkrss krapqmdwsk knelfsnl
The amino acid sequence of the human form of Cbp/PAG appears in SEQ ID No . 4 : mgpagsllgs gqmqitlwgs laavaiffvi tflifpcssc drekkprqhs gdhenlmnvp1 sdkemfsrsv tslatdapas seqngaltng dilsedstlt cmqhyeevqt sasdlldsqd 1 stgkpkchqs relprippes avdtmltars vdgdqglgme gpyevlkdss sqenmvedcl81 yetvkeikev aaaahlekgh sgkakstsas kelpgpqteg kaefaeyasv drnkkcrqsv 1 nvesilgnsσ dpeeeapppv pvklldenen lqekeggeae esatdttset nkrfsslsyk 01 sreedptlte eeisamyssv nkpgqlvnks gqsltvpest ytsiqgdpqr spsscndlya61 tvkdfektpn stlppagrps eepepdyeai qtlnreeeka tlgtnghhgl vpkendyesi21 sdlqqgrdit rl
Preferably the binding partners ezrin, PKAI, EBP50 and Cbp/PAG as described herein refer to a polypeptide comprising SEQ ID NO . 1, 2, 3 and 4, respectively, and their functionally equivalent variants, derivatives or fragments. Especially preferably the binding partners are those molecules which occur endogenously . Such variants, derivatives and fragments are described hereinafter with particular reference to inhibitors of the invention. Variants, derivatives and fragments of the binding partners as mentioned herein are similarly defined. In particular such variants include naturally occurring variants such as comparable proteins found in other species or more particularly variants and alleles found within humans. Conveniently, said variants may be described as having more than 75%, e.g. 80, 85 or 90, especially preferably more than 95% sequence similarly or identity to the sequence described in SEQ ID No. 1, 2, 3 or 4. Thus in a preferred aspect, the present invention provides a method of altering the PKA type I signalling pathway in a cell by administration of an inhibitor (or a molecule encoding an inhibitor) as defined herein, which reduces or inhibits binding between one or more of the following binding partners : i) a polypeptide comprising the sequence as set forth in SEQ ID No. 1 or a sequence with 95% similarity thereto or a sequence encoded by a nucleotide sequence which hybridizes under conditions of high stringency to the nucleotide sequence encoding the amino acid sequence of SEQ ID No. 1, or a functionally equivalent fragment thereof, and a polypeptide comprising the sequence as set forth in SEQ ID No. 2 or a sequence with 95% similarity thereto or a sequence encoded by a nucleotide sequence which hybridizes under conditions of high stringency to the nucleotide sequence encoding the amino acid sequence of SEQ ID No . 2 , or a functionally equivalent fragment thereof; ii) a polypeptide comprising the sequence as set forth in SEQ ID No. 1 or a sequence with 95% similarity thereto or a sequence encoded by a nucleotide sequence which hybridizes under conditions of high stringency to the nucleotide sequence encoding the amino acid sequence of SEQ ID No . 1, or a functionally equivalent fragment thereof, and a polypeptide comprising the sequence as set forth in SEQ ID No. 3 or a sequence with 95% similarity thereto or a sequence encoded by a nucleotide sequence which hybridises under conditions of high stringency to the nucleotide sequence encoding the amino acid sequence of SEQ ID No . 3 , or a functionally equivalent fragment thereof; iii) a polypeptide comprising the sequence as set forth in SEQ ID No. 3 or a sequence with 95% similarity thereto or a sequence encoded by a nucleotide sequence which hybridizes under conditions of high stringency to the nucleotide sequence encoding the amino acid sequence of SEQ ID No. 3, or a functionally equivalent fragment thereof, and a polypeptide comprising the sequence as set forth in SEQ ID No. 4 or a sequence with 95% similarity thereto or a sequence encoded by a nucleotide sequence which hybridizes under conditions of high stringency to the nucleotide sequence encoding the amino acid sequence of SEQ ID No . 4, or a functionally equivalent fragment thereof. In connection with amino acid sequences, "sequence similarity", preferably "identity", refers to sequences which have the stated value when assessed using e.g. using the SWISS-PROT protein sequence databank using FASTA pep-cmp with a variable pamfactor, and gap creation penalty set at 12.0 and gap extension penalty set at 4.0, and a window of 2 amino acids) . Sequence identity at a particular residue is intended to include identical residues which have simply been derivatized. "Hybridizing under conditions of high stringency" refers to hybridization under non-stringent binding conditions of 6 x SSC/50% formamide at room temperature and washing under conditions of high stringency, e.g. 2 x SSC, 65°C, where SSC = 0.15 M NaCl, 0.015M sodium citrate, pH 7.2. As referred to herein, sequences encoded by a sequence which hybridizes to a particular sequence refers to the polypeptide sequence encoded by the complementary sequence of a sequence which hybridizes to the particular sequence. Alternatively, or additionally, such hydridizing sequences may be described as those which exhibit at least 70%, preferably at least 80 or 90%, e.g. at least 95% sequence identity (as determined by, e.g. FASTA Search using GCG packages, with default values and a variable pamfactor, and gap creation penalty set at 12.0 and gap extension penalty set at 4.0 with a window of 6 nucleo ides) to the sequence which encodes the recited polypeptide, or a sequence complementary to any of the aforesaid sequences, or a fragment of any of the aforesaid sequences encoding the relevant binding region. Especially preferably said binding partners consist of, or comprise, specific fragments (functionally equivalent fragments) of said above described polypeptides which correspond to the relevant binding sites . Ezrin binds to PKA type I through bases in the region of 356 to 470 (particularly in the region 363 to 470) . Within this region, residues 413 or 414 to 443 (containing the amphipathic helix binding domain at residues 413-430) are involved in binding to PKA type I. A second region has also been identified that provides specificity and additional affinity in anchoring the enzyme. This region is found at residues 356 to 397 (particularly 367 to 390) and is described in more detail in Example 3 and Figure 10. This region has been found to comprise two separate binding elements, namely from residues 356 to 376 and 378 to 397 (see Example 4) . Binding to PKA involves at least residues 1-50 of that enzyme. Residues 1-286 of ezrin bind to residues 329-358 of EBP50. Residues 11-97 and/or 149-236 of EBP50 bind to the C-terminal 4 amino acids of Cbp/PAG. Thus, in a particularly preferred aspect said binding partner polypeptide consists of at least the following amino acid sequence or a sequence with 95% similarity thereto or a sequence encoded by a nucleotide sequence which hybridizes under conditions of high stringency thereto: (a) amino acids 356 to 470, (e.g. 363-470) (for binding to SEQ ID No. 2 or its variants) or 1 to 286 (for binding to SEQ ID No . 3 or its variants) of SEQ ID No. 1; (b) amino acids 1 to 50 of SEQ ID No . 2 (for binding to SEQ ID No. 1 or its variants) ; (c) amino acids 11-97 (for binding to SEQ ID No . 4 or its variants), 149-236 (for binding to SEQ ID No. 4 or its variants) or 329-358 (for binding to SEQ ID No. 1 or its variants) of SEQ ID No. 3; (d) amino acids 429 to 432 of SEQ ID No . 4 (for binding to SEQ ID No . 3 or its variants) . Where appropriate, inhibitors of the binding between even smaller fragments of the afore-described binding partners may be used. Binding between these binding partners may be affected in a variety of ways. Conveniently inhibitors of said binding which directly interfere with the binding at the binding site may be employed. Alternatively and as described hereinafter, binding may be reduced by modifying the endogenous molecules taking part in binding. Both inhibitors and the molecules used to achieve an alteration in the form or expression of one or more of the endogenous binding partners are referred to herein as inhibitors even though in the latter case their mode of interaction is not necessarily to achieve direct inhibition of the binding site (ie. a steric inhibitor) . Thus molecules which act to ultimately achieve inhibition of binding between binding partners (preferably endogenous binding partners) are referred to herein as inhibitors . Such molecules include peptides and proteins as well as nucleic acid molecules, such as those which encode peptide/protein inhibitors which may be used to express the peptide/protein inhibitors within the cell or may be used to derive sense of antisense nucleotide sequences to cause co-suppression or suppression to reduce expression of the endogenous binding partner. Thus in a further aspect the present invention provides an inhibitor molecule which reduces or inhibits the binding between one or more of the binding partners as defined hereinbefore and which preferably is capable of altering the PKA type I signalling pathway in a cell. Preferably direct inhibitors of binding are antagonists of binding between the specific binding pairs . Such inhibitors may themselves bind to one of the binding pair at the binding site (e.g. as described hereinbefore) or at a site on one or other of the binding partners which prevents the successful interaction of those binding partners, e.g. through steric interference or altering the properties of the binding site, e.g. its spatial or charge conformation. As referred to herein an "antagonist" is a molecule or complex of molecules which by virtue of structural similarity to one molecule of a binding pair competes with that molecule for binding to the other molecule of the binding pair. Thus the inhibitors may be molecules which specifically recognize the binding site, such as antibodies (or fragments thereof) , or proteins or peptides which associate with that region or associate sufficiently close to affect accessibility by the other binding partner. Alternatively, molecules, particularly peptides or larger molecules, which mimic the binding site (or contain a region which mimics the binding site) (e.g. peptides, proteins or anti-idiotypes) may be used to act instead as the pseudo-binding partner to reduce the extent of binding between the true endogenous binding partners . Preferred mimics are peptides that comprise the relevant binding site as described herein. Molecules which are complementary to the binding site, e.g. such that they bind to that site may also be used. Molecules which affect the binding site indirectly, by binding at a distant part of the molecule, but which affect the conformation of the binding site and thus the ability to form relevant interactions with its binding partner, may also be used. Appropriate molecules may for example be proteins or peptides or other molecules which can affect binding, or a nucleic acid molecule which encodes such a product may be used to generate the inhibitor. Conveniently small inhibitor molecules may be used. However, where desired, large molecules containing the binding site may be employed, e.g. larger molecules which mimic the entire binding partner (e.g. the soluble portion thereof) , but which have preferably been modified such that one or more properties required for involvement in the signalling pathway is missing or altered to lose or impair its ability to be involved in signalling. For example in the case of EBP50 a large molecule absent only the 30 C-terminal amino acids may be used, which would allow functional binding with Cbp/PAG, but not ezrin. Alternatively whilst the ability to bind one binding partner may be retained, other relevant binding sites required to allow the development of the signalling scaffold may be impaired or removed such that a functional complex is not formed, e.g. by mutation. For example, in the case of ezrin, the full length molecule could be used with a mutation at the binding site for either EBP50 or PKA type I. Thus the present invention further extends to an inhibitor which is a polypeptide (or the nucleic acid molecules encoding it) containing one or more mutations in one or more binding sites described herein, in which said mutation results in a molecule which has impaired binding at that site to the relevant binding partner relative to the same molecule without the mutation. For example, one or more of the valine and/or isoleucine residues in the PKA binding site of the ezrin molecule, ie. amino acids 356 or 363 to 470 of SEQ ID No. 1 may be mutated, preferably to a proline residue, and the resultant molecule used as an inhibitor to interfere with the ability of the endogenous enzyme to bind to its binding partner PKA. The present invention thus also extends to novel modified, e.g. mutated binding partners or functionally equivalent variants, derivatives or fragments thereof and the nucleic acid molecules which encode them. Thus, viewed from a yet further aspect, the present invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding a binding partner, or a functionally equivalent variant, derivative or fragment thereof, as defined above, wherein said sequence is modified as defined above to alter its ability to bind to its binding partner as defined above. Appropriate inhibitors for use in methods of the invention may be identified or tested using appropriate screening tests. Thus in a further aspect, the present invention provides a method of screening for, or testing the ability or efficacy of, a molecule to reduce or inhibit binding between any one of the aforementioned binding partners, wherein said test molecule is contacted with said binding partners and the extent of binding is assessed. Optionally said binding partners are present in isolated form, for example the first of said binding pair may be immobilized on a solid support and the ability of the second of said pair to bind to said first binding partner may be assessed in the presence or absence of said test molecule, ie. by competition. Alternatively, said binding partners may be present in endogenous form, e.g. in a cell and the ability of said test molecule to affect PKA type I signalling by examination of an indicator of said signalling, e.g. T-cell proliferation or IL-2 release, may be examined. Preferred inhibitors of binding between the ezrin and PKA type I binding partners as described hereinbefore mimic the PKA type I binding site on ezrin, ie. residues 356-470 or 363-470, e.g. 356-397, 363-390, 397-470 or 390-470 of SEQ ID No. 1, e.g. residues 356- 376, 363-404, 363-412, 367-390, 378-397, 390-412, 413- 420, 413-430, 414-443 and/or 421-470 or a variant thereof as described hereinafter. Preferably the inhibitors are derived from and/or mimic said binding site. For example, said inhibitor may comprise or may encode one or more of the residues mentioned above and/or e.g. 363-382, 366-385, 369-388, 372-391, 375-394, 378-397, 381-400, 390-409, 393-412, 414-433, 416-435, 417-436, 418-437, 420-439, 422-441, 424-443, 429-448, 432-451, 435-454 and 441-460 of SEQ ID No. 1 or functionally equivalent variants, derivatives or fragments of such peptides as described hereinafter. Preferably said inhibitors as described herein consist of less than 100 amino acids, e.g. are or encode peptides which consist of less than 100 amino acids, but are preferably shorter, e.g. less than 50, e.g. less than 30 residues in length, e.g. from 8 to 20 amino acids, e.g. from 10 to 15 residues. "Functionally equivalent" variants, derivatives or fragments thereof refers to molecules, preferably peptides, related to, or derived from the above described amino acid sequences, where the amino acid sequence has been modified by single or multiple amino acid (e.g. at 1 to 10, e.g. l to 5, preferably 1 or 2 residues) substitution, addition and/or deletion or chemical modification, including deglycosylation or glycosylation, but which nonetheless retain functional activity, insofar as they act as inhibitors, e.g. as binding partner mimics and thus antagonize the interaction between the binding partners (or in the case of functionally equivalent binding partners retain the ability to bind to their respective binding partners) . Within the meaning of "addition" variants are included amino and/or carboxyl terminal fusion proteins or polypeptides, comprising an additional protein or polypeptide or other molecule fused to the inhibitor (or binding partner) sequence. "Substitution" variants preferably involve the replacement of one or more amino acids with the same number of amino acids and making conservative substitutions . Such functionally-equivalent variants mentioned above include in particular naturally occurring biological variations (e.g. allelic variants or geographical variations within a species, most particularly variants and alleles found within humans) and derivatives prepared using known techniques. In particular functionally equivalent variants of the inhibitors (or binding partners) described herein extend to inhibitors (or binding partners) which are functional in (or present in) , or derived from proteins isolatable from, different genera or species than the specific inhibitors and binding partner molecules mentioned herein. Preferred "derivatives" or "variants" include those in which instead of the naturally occurring amino acid the amino acid which appears in the sequence is a structural analog thereof . Amino acids used in the sequences may also be derivatized or modified, e.g. labelled, glycosylated or methylated, providing the function of the inhibitor (or binding partner) is not significantly adversely affected. Derivatives particularly include peptidomimetics which may be prepared using techniques known in the art. For example, non-standard amino acids such as ot- aminobutyric acid, penicillamine, pyroglutamic acid or conformationally restricted analogs, e.g. such as Tic (to replace Phe) , Aib (to replace Ala) or pipecolic acid (to replace Pro) may be used. Other alterations may be made when the inhibitor is to be used in the methods of the invention described hereinafter (or the binding partners are to be used for screening for inhibitors) . In such cases, the stability of the inhibitor (or binding partner), e.g. peptide, may be enhanced, e.g. by the use of D-amino acids, or amide isosteres (such as N- methyl amide, retro-inverse amid, thioamide, thioester, phosphonate, ketomethylene, hydroxymethylene, fluorovinyl, (E) -vinyl, methyleneamino, methylenethio or alkane) which protect the peptides against proteolytic degradation. Di (oligo) peptidomimetics may also be prepared. Precursors of the inhibitors (or binding partners) are also encompassed by the term functionally equivalent variants and include molecules which are larger than the inhibitors (or binding partners) and which may optionally be processed, e.g. by proteolysis to yield the inhibitor (or binding partner) . Additional moieties may also be added to the inhibitors (or binding partners) to provide a required function, e.g. a moiety may be attached to assist or facilitate entry of the inhibitor into the cell . Derivatives and variants as described above may be prepared during synthesis of the inhibitor (or binding partner if isolated binding partners are to be used) , e.g. peptide, or by post-production modification, or when the peptide is in recombinant form, using the known techniques of site-directed mutagenesis including deletion, random mutagenesis and/or ligation of nucleic acids . Functionally-equivalent "fragments" according to the invention may be made by truncation, e.g. by removal of a peptide from the N and/or C-terminal ends. Such fragments may be derived from the inhibitor peptides (or binding partners) described above, or may be derived from a functionally equivalent peptide as described above, but which retain the ability to act as an inhibitor (or binding partner) according to the method of the invention. Preferably such fragments are between 6 and 30 residues in length, e.g. 6 to 25 or 10 to 15 residues. Preferably these fragments satisfy the homology (relative to a comparable region) or hybridizing conditions mentioned herein. Preferably functional variants according to the invention have an amino acid sequence which has more than 75%, e.g. 75 or 80%, preferably more than 85%, e.g. more than 90 or 95% or 98% similarity or identity to the aforementioned inhibitor or binding partner sequences (according to the test described hereinbefore) . Especially preferably said nucleotide sequences are the degenerate sequences which encode the recited polypeptides or their variants or fragments . Especially preferably peptide inhibitors in this aspect (ezrin: PKA binding) comprise the following sequence or a functionally equivalent variant, derivative or fragment thereof, based on residues 413- 430:
SQEX1LAX2X3LAX4X5XSX7KX8AX9
wherein each of Xi to Xs is any amino acid, wherein Xi is preferably arginine, glutamic acid, glutamine, threonine, phenylalanine, lysine or tyrosine, especially preferably glutamine; X2 is preferably arginine, lysine, glycine, phenylalanine, alanine, serine or asparagine, especially preferably alanine; X3 is preferably arginine, glutamic acid, glutamine, threonine, lysine, serine, valine, alanine or leucine, especially preferably glutamic acid; X4 is preferably glutamic acid, glutamine, lysine, leucine, serine, aspartic acid, alanine, glycine or asparagine, especially preferably glutamic acid; X5 is preferably tyrosine; Xs is preferably threonine, valine, isoleucine, alanine or serine, especially preferably threonine; and wherein X7 is any amino acid other than proline, preferably valine, alanine, isoleucine or leucine, preferably isoleucine or alanine, especially preferably alanine; X8 is any amino acid other than proline, preferably isoleucine, glutamic acid or glutamine, especially preferably isoleucine; and X is any amino acid other than proline, preferably leucine .
In a preferred aspect, said sequence is:
SQEXiLAXzXgLAXa XβAKIA .
Alternatively said sequence may be based on ezrin residues 367-385 and may have the form: D/EX1X2X3X4X5LX6X7X8EEX9X;L0X11X.L2X13EE
wherein each of X_ and X2 is any amino acid, wherein X± is preferably arginine, glutamine, alanine, glutamic acid or isoleucine, preferably glutamine; X2 is preferably asparagine, isoleucine, glutamic acid or glutamine, preferably isoleucine; and wherein X3 is glutamic acid, glutamine, valine, arginine or phenylalanine, preferably glutamine; X4 is glutamic acid, arginine, leucine or methionine, preferably arginine; Xs is glycine, alanine, arginine or glutamic acid, preferably alanine,- X6 is aspartic acid, glutamine, lysine or arginine, preferably glutamine; X7 is arginine, leucine, methionine or lysine, preferably leucine; XB is asparagine, glutamic acid, alanine or methionine, preferably glutamic acid; X9 is serine, arginine, lysine or leucine, preferably arginine; X10 is leucine, lysine, glutamic acid, arginine or valine, preferably lysine; X__ is aspartic acid, arginine or lysine, preferably arginine; X12 is arginine, alanine or phenylalanine, preferably alanine; and X13 is asparagine, glutamine, alanine, glutamic acid or tryptophan, preferably glutamine.
In a preferred aspect, said sequence is : D/ EXIX2X3X4ALQX7EEEX X^QRXI XI3EE .
Such inhibitors or their encoding nucleic acid sequences form further preferred aspects of the invention for use in methods of the invention such as a method of altering the PKA type I signalling pathway in a cell by administration of said inhibitor. Preferred inhibitors of binding between the ezrin and EBP50 binding partners as described hereinbefore mimic the ezrin binding site on EBP50 or the EBP50 binding site on ezrin, ie. residues 329 to 358 of SEQ ID No. 3 (EBP50) , or residues 1-286 of SEQ ID No. 1 (ezrin) , respectively, or variants as described hereinbefore . Preferably the inhibitors are derived from and/or mimic said binding sites. For example, said inhibitor may comprise or encode one or more of residues 1-20, 20- 40, 40-60, 60-80, 80-100, 100-120, 120-140, 140-160, 160-180, 180-200, 200-220, 220-240, 240-260 or 260-286 of SEQ ID No. 1 or 329 to 349 or 339 to 358 of SEQ ID No. 3 or functionally equivalent variants, derivatives or fragments of such peptides as described hereinbefore. Preferred inhibitors of binding between the EBP50 and Cbp/PAG binding partners as described hereinbefore mimic the EBP50 binding site on Cbp/PAG or the Cbp/PAG binding site on EBP50, ie . 429 to 432 residues of SEQ ID No. 4 (Cbp/PAG) , or residues 11 to 97 or 149 to 236 of SEQ ID No . 3 (EBP50) , respectively, or variants as described hereinbefore. Preferably the inhibitors are derived from and/or mimic said binding sites. For example, said inhibitor may comprise or encode one or more of residues 429-432 of SEQ ID No. 4 or residues 11-30, 31-50, 51-70, 71-90, 80-97, 149-170, 171-190, 191-210, 211-230 or 215-236 of SEQ ID No. 3 or functionally equivalent variants, derivatives or fragments of such peptides as described hereinbefore . As mentioned previously, said above described inhibitors may where appropriate include full length molecules identical or closely related to those found endogenously to compete with the wild-type molecule. As such, appropriate inhibitors include any of the aforementioned binding partner proteins in complete form. The present invention also extends to antibodies (monoclonal or polyclonal) and their antigen-binding fragments (e.g F(ab)2, Fab and Fv fragments ie. fragments of the "variable" region of the antibody, which comprises the antigen binding site) directed to the binding sites or inhibitor peptides as defined hereinbefore, ie . which bind to epitopes present on the peptides or binding sites and thus bind selectively and specifically to such peptides or binding sites and which may be used to inhibit the binding of the binding partners . Inhibitors as described herein are conveniently proteinaceous, however the use of other types of inhibitors of the binding is contemplated, e.g. molecules which match the binding sites' spatial and conformational structure. In order to affect the signalling pathway, inhibitor molecules as described hereinbefore are conveniently added to a cell . This may be achieved by relying on spontaneous uptake of the inhibitor into the cells or appropriate carrier means may be provided. Exogenous peptides or proteins may thus be introduced by any suitable technique known in the art such as in a liposome, niosome or nanoparticle or attached to a carrier or targetting molecule (see hereinafter) . Thus for example, the protein may be tagged with a suitable sequence that allows the protein to cross the cell membrane. An example of such a tag is the HIV tat sequence or a stretch of e.g. 11 arginines . It will be appreciated that the level of exogenous molecules introduced into a cell will need to be controlled to avoid adverse effects . The inhibitor may be transported into the cell in the form of the inhibitor or in the form of a precursor, e.g. with an attached moiety to allow passage across the cell membrane or for cell targeting or in a form which is only activated on conversion, e.g. by proteolysis or transcription and translation. In the case of peptide or protein inhibitors, the inhibitors may be administered to a cell by transfection of a cell with a nucleic acid molecule encoding the peptide or protein inhibitor. The present invention thus extends to nucleic acid molecules comprising a sequence which encodes the peptide/polypeptide inhibitors described herein and their use in methods described herein. Preferably said nucleic acid molecules are contained in a vector, e.g. an expression vector. The nucleic acid molecules described above may be operatively linked to an expression control sequence, or a recombinant DNA cloning vehicle or vector containing such a recombinant DNA molecule. This allows intracellular expression of the inhibitor as a gene product, the expression of which is directed by the gene(s) introduced into cells of interest. Gene expression is directed from a promoter active in the cells of interest and may be inserted in any form of linear or circular DNA vector for incorporation in the genome or for independent replication or transient transfection/expression. Alternatively, the naked DNA molecule may be injected directly into the cell. Appropriate expression vectors include appropriate control sequences such as for example translational (e.g. start and stop codons, ribosomal binding sites) and transcriptional control elements (e.g. promoter- operator regions, termination stop sequences) linked in matching reading frame with the nucleic acid molecules required for performance of the method of the invention. Appropriate vectors may include plasmids and viruses (including both bacteriophage and eukaryotic viruses) . Suitable viral vectors include baculovirus and also adenovirus, adeno-associated virus, herpes and vaccinia/pox viruses . Many other viral vectors are described in the art. Preferred vectors include bacterial and mammalian expression vectors pGEX-KG, pEF- neo and pEF-HA. The nucleic acid molecule may conveniently be fused with DNA encoding an additional polypeptide, e.g. glutathione-S-transferase, to produce a fusion protein on expression. Thus viewed from a further aspect, the present invention provides a vector comprising a nucleic acid molecule as defined above. Other aspects of the invention include methods for preparing recombinant nucleic acid molecules according to the invention, comprising inserting nucleotide sequences encoding the modified substrate into vector nucleic acid. Nucleic acid molecules of the invention, preferably contained in a vector, may be introduced into a cell by any appropriate means . Suitable transformation or transfection techniques are well described in the literature. A variety of techniques are known and may be used to introduce such vectors into prokaryotic or eukaryotic cells for expression. Preferred host cells for this purpose include insect cell lines, eukaryotic cell lines or E. coli , such as strain BL21/DE3. The invention also extends to transformed or transfected prokaryotic or eukaryotic host cells containing a nucleic acid molecule, particularly a vector as defined above. A further aspect of the invention provides a method of preparing a peptide or protein inhibitor of the invention as hereinbefore defined, which comprises culturing a host cell containing a nucleic acid molecule as defined above, under conditions whereby said inhibitor is expressed and recovering said inhibitor thus produced. The expressed protein product forms a further aspect of the invention. The invention also extends to an inhibitor encoded by a nucleic acid molecule as hereinbefore described. This may be produced by expression of a host cell as described above. Cells containing inhibitors of the invention, introduced directly as inhibitors or by expression of encoding nucleic acid material form further aspects of the invention. Nucleic acid molecules which may be used according to the invention may be single or double stranded DNA, cDNA or RNA, preferably DNA and include degenerate, substantially homologous and hybridizing sequences as described before. Ideally however genomic DNA or cDNA is employed. Inhibitors as described above may be prepared by conventional modes of synthesis including genetic or chemical means . Chemical syntheses may be performed by methods well known in the art involving in the case of peptides cyclic sets of reactions of selection deprotection of the functional groups of a terminal amino acid and coupling of selectively protected amino acid residues, followed finally by complete deprotection of all functional groups. Synthesis may be performed in solution or on a solid support using suitable solid phases known in the art, such as the well known Merrifield solid phase synthesis procedure. Preferably the inhibitors of the invention are substantially purified, e.g. pyrogen-free, e.g. more than 70%, especially preferably more than 90% pure (as assessed for example, in the case of peptides or proteins, by an appropriate technique such as peptide mapping, sequencing or chromatography) . Purification may be performed for example by chromatography (e.g. HPLC, size-exclusion, ion-exchange, affinity, hydrophobic interaction, reverse-phase) or capillary electrophoresis . Alternative methods of reducing binding between the binding partners as defined hereinbefore includes modification of endogenous molecules taking part in said binding. Thus, the invention extends to modifying the endogenous binding partner as described hereinbefore in a cell . This may be achieved for example by manipulation of the wild-type gene, by manipulating expression of the gene (e.g. by affecting transcription or translation) or by manipulating the expressed product. This could for example be achieved by using antisense oligonucleotides comprising nucleic acid sequences as described hereinbefore (ie. of binding partners or of relevant protein/peptide inhibitors) or their complementary sequences, ribozymes, RNAi or antibodies and the invention extends to such molecules and their uses. For example, to manipulate the endogenous gene, this could be performed for example by somatic cell gene therapy with homologous recombination to for example remove or mutate the binding site. This could be performed on for example hematopoietic stem cells or on blood cells ex vivo or in vivo . Mutation of one or more of the leucine or valine residues to a proline residue in the ezrin binding site for PKA for example could be performed to generated proteins that have reduced binding to PKA. Alternatively wild-type or mutated sequences may be used to cause co-suppression of the naturally occurring molecule. Such exogenous molecules may be administered to cells as described hereinbefore. The above described methods of the invention may be used in vi tro, for example in cell or organ culture, particularly for affecting PKA type I signalling pathways which have been activated or to reduce the extent of endogenous signalling. The method may also be used ex vivo, on animal parts or products, for example organs or collected blood, cells or tissues, particularly when it is contemplated that these will be reintroduced into the body from which they are derived. In particular, in samples in which abnormal levels of PKA type I signalling are occurring, levels may be normalized, e.g. by inhibiting the activity of the PKA type I signalling pathway. In such a method of treatment, the sample may be harvested from a patient and then returned to that patient . In this context, a "sample" refers to any material obtained from a human or non-human animal, including tissues and body fluid. "Body fluids" in this case include in particular blood, spinal fluid and lymph and "tissues" include tissue obtained by surgery or other means . Such methods are particularly useful when the inhibitor is to be introduced into the body by expression of an appropriate nucleic acid molecule, or the inhibitor is itself a nucleic acid molecule. T cells for example, could be treated in this way. In such methods the methods of treatment of the invention as described hereinafter comprise the initial step of obtaining a sample from an individual, contacting cells from said sample with an inhibitor (or a nucleic acid molecule encoding an inhibitor) of the invention and administering said cells of said sample to the individual . The step of contacting refers to the use of any suitable technique which results in the presence of said inhibitor in cells of the sample. The method may also be used in vivo for the treatment or prevention of diseases in which abnormal PKA type I signalling occurs and this will be discussed in more detail below. As described previously the methods of altering PKA type I signalling have utility in a variety of clinical indications in which abnormal PKA type I signalling is exhibited. In particular since PKA type I is a key negative regulator of T cell function, diseases which exhibit lymphocyte dysfunction are particular targets for this treatment. Specifically, the inhibitors which abolish the function of PKA type I may be used to produce pharmaceutical preparations to treat immunosuppressive disease. Thus, the inhibitors may be used to treat or prevent disorders typified by aberrant PKA type I signalling such as immunosuppressive disorders (such as HIV infection, AIDS or common variable immunodeficiency) or proliferative diseases in which PKA type I signalling has been implicated, e.g. cancers such as colorectal carcinoma, pancreatic carcinoma, hepatocellular carcinoma, cancer mamma, ovarian cancer and non-small cell carcinoma of the lung) . The inhibitors as described herein may therefore be formulated as pharmaceutical compositions in which the inhibitor may be provided as a pharmaceutically acceptable salt. Pharmaceutically acceptable salts may be readily prepared using counterions and techniques well known in the art. The invention thus further extends to pharmaceutical compositions comprising one or more inhibitors (e.g. nucleic acid molecules, peptides or proteins, such as an antisense oligonucleotide, ribozyme or antibody, nucleic acid molecule or peptide/protein) as defined above and one or more pharmaceutically acceptable excipients and/or diluents . By "pharmaceutically acceptable" is meant that the ingredient must be compatible with other ingredients in the composition as well as physiologically acceptable to the recipient. The active ingredient for administration may be appropriately modified for use in a pharmaceutical composition. For example when peptides are used these may be stabilized against proteolytic degradation by the use of derivatives such as peptidomimetics as described hereinbefore. The active ingredient may also be stabilized for example by the use of appropriate additives such as salts or non-electrolytes, acetate, SDS, EDTA, citrate or acetate buffers, mannitol, glycine, HSA or polysorbate. Conjugates may be formulated to provide improved lipophilicity, increase cellular transport, increase solubility or allow targeting. Conjugates may be made terminally or on side portions of the molecules, e.g. on side chains of amino acids. These conjugates may be cleavable such that the conjugate behaves as a pro-drug. Stability may also be conferred by use of appropriate metal complexes, e.g. with Zn, Ca or Fe . The active ingredient may be formulated in an appropriate vehicle for delivery or for targeting particular cells, organs or tissues. Thus the pharmaceutical compositions may take the form of microemulsions, liposomes, niosomes or nanoparticles with which the active ingredient may be absorbed, adsorbed, incorporated or bound. This can effectively convert the product to an insoluble form. These particulate forms have utility for transfer of nucleic acid molecules and/or protein/peptides and may overcome both stability (e.g. enzymatic degradation) and delivery problems . These particles may carry appropriate surface molecules to improve circulation time (e.g. serum components, surfactants, polyoxamine908 , PEG etc.) or moieties for site-specific targeting, such as ligands to particular cell borne receptors . Appropriate techniques for drug delivery and for targeting are well known in the art and are described in W099/62315. For an example of specific site directed targeting, see for example Schafer et al . , 1992, Pharm. Res., 9, p541-546 in which nanoparticles can be accumulated in HIV-infected macrophages. Clearly such methods have particular applications in the methods of the invention described herein. Such derivatized or conjugated active ingredients are intended to fall within the definition of inhibitory molecules which form aspects of this invention. Pharmaceutical compositions for use according to the invention may be formulated in conventional manner using readily available ingredients. Thus, the active ingredient may be incorporated, optionally together with other active substances as a combined preparation, with one or more conventional carriers, diluents and/or excipients, to produce conventional galenic preparations such as tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions (as injection or infusion fluids) , emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium) , ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions, sterile packaged powders, and the like. Biodegradable polymers (such as polyesters, polyanhydrides , polylactic acid, or polyglycolic acid) may also be used for solid implants. The compositions may be stabilized by use of freeze-drying, undercooling or Permazyme . Suitable excipients, carriers or diluents are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, calcium carbonate, calcium lactose, corn starch, aglinates, tragacanth, gelatin, calcium silicate, macrocrystalline cellulose, polyvinylpyrrolidone, cellulose, water syrup, water, water/ethanol, water/glycol, water/polyethylene, glycol, propylene glycol, methyl cellulose, methylhydroxybenzoates, propyl hydroxybenzoates, talc, magnesium stearate, mineral oil or fatty substances such as hard fat or suitable mixtures thereof. Agents for obtaining sustained release formulations, such as carboxypolymethylene, carboxymethyl cellulose, cellulose acetate phthalate, or polyvinylacetate may also be used. The compositions may additionally include lubricating agents, wetting agents, viscosity increasing agents, colouring agents, granulating agents, disintegrating agents, binding agents, osmotic active agents, emulsifying agents, suspending agents, preserving agents, sweetening agents, flavouring agents, adsorption enhancers, e.g. for nasal delivery (bile salts, lecithins, surfactants, fatty acids, chelators) and the like. The compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration of the patient by employing procedures well known in the art . The active ingredient in such compositions may comprise from about 0.01% to about 99% by weight of the formulation, preferably from about 0.1 to about 50%, for example 10%. The invention also extends to pharmaceutical compositions as described above for use as a medicament. In methods of the invention, inhibitors should be used at appropriate concentrations such that a significant number of the relevant binding partners' interactions, are prevented. Preferably the pharmaceutical composition is formulated in a unit dosage form, e.g. with each dosage containing from about 0.1 to 500mg of the active ingredient. The precise dosage of the active compound to be administered and the length of the course of treatment will of course, depend on a number of factors including for example, the age and weight of the patient, the specific condition requiring treatment and its severity, and the route of administration. Generally however, an effective dose may lie in the range of from about O.Olmg/kg to 20mg/kg, depending on the animal to be treated, and the substance being administered (e.g 0.1 to 7mg/kg for antisense oligonucleotides) , taken as a single dose. For methods in which the inhibitor or its encoding molecule is administered to a sample ex vivo to be returned to the body, suitable dosages of said inhibitor are 25-100nM or lower, such as 10-50nM or 5-25nM. The administration may be by any suitable method known in the medicinal arts, including for example oral, parenteral (e.g. intramuscular, subcutaneous, intraperitoneal or intravenous) percutaneous, buccal, rectal or topical administration or administration by inhalation. The preferred administration forms will be administered orally, rectally or by injection or infusion. As will be appreciated oral administration has its limitations if the active ingredient is digestible. To overcome such problems, ingredients may be stabilized as mentioned previously and see also the review by Bernkop-Schnύrch, 1998, J. Controlled Release, 52, pl-16. It will be appreciated that since the active ingredient for performance of the invention takes a variety of forms, e.g. oligonucleotide, antibody, ribozyme, nucleic acid molecule (which may be in a vector or host cell) or peptide/protein, the form of the composition and route of delivery will vary. Preferably however liquid solutions or suspensions would be employed, particularly e.g. for nasal delivery and administration will be systemic. As mentioned above, these pharmaceutical compositions may be used for treating or preventing conditions in which the PKA type I signalling pathway is abnormal, in particular when the activity of this pathway is elevated. Thus, viewed from a further aspect the present invention provides a method of treating or preventing disorders exhibiting abnormal PKA type I signalling activity, preferably immunosuppressive disorders or proliferative diseases, in a human or non-human animal wherein a pharmaceutical composition as described hereinbefore is administered to said animal . Alternatively stated, the present invention provides the use of a pharmaceutical composition as defined above for the preparation of a medicament for the treatment or prevention of immunosuppressive disorders or proliferative diseases. As referred to herein a "disorder" or "disease" refers to an underlying pathological disturbance in a symptomatic or asymptomatic organism relative to a normal organism, which may result, for example, from infection or an acquired or congenital genetic imperfection. A "condition" refers to a state of the mind or body of an organism which has not occurred through disease, e.g. the presence of a moiety in the body such as a toxin, drug or pollutant. As referred to herein an "immunosuppressive disorder" refers to a disorder which is typified by impaired function of cells involved in normal immune responses, particularly B and T cells, and is also referred to herein as immunodeficiency or immune dysfunction. Preferably virally-induced immunodeficiency disorders are treated. Preferred conditions for treatment according to the invention include infection by retroviruses, particularly HIV (and infection by related viruses in other animals, e.g. SIV, FIV, MAIDS) and the resultant AIDS and treatment of common variable immunodeficiency and related conditions to the aforementioned conditions. Especially preferably the methods described herein may be used to reverse cAMP hyperactivation that produces T cell dysfunction in immunodeficiencies by interrupting cAMP and PKA-mediated signal transduction in T cells, particularly T cell lipid rafts. As referred to herein "proliferative diseases" refers to those diseases in which aberrant proliferation of cells occurs. Preferably said cells are cells involved in the generation or maintenance of immune responses, particularly B or T cells. Such diseases concern decreases in proliferation relative to normal levels . Subjects which may be treated are preferably mammalian, preferably humans and companion or agricultural animals such as dogs, cats, monkeys, horses, sheep, goats, cows, rabbits, rats and mice. As used herein, "treating" refers to the reduction, alleviation or elimination, preferably to normal levels, of one or more of the symptoms of said disease, disorder or condition which is being treated, e.g. infectivity or a reduction or alleviation of immune dysfunction, relative to the symptoms prior to treatment. "Preventing" refers to absolute prevention, ie . absence of detectable infectious agents, e.g. virus and/or maintenance of normal levels with reference to the extent or appearance of a particular symptom (e.g. T cell numbers) or reduction or alleviation of the extent or timing (e.g. delaying) of the onset of that symptom. The method of treatment according to the invention may advantageously be combined with administration of one or more active ingredients which are effective in treating the disorder or disease to be treated. Preferably such additional active ingredients are cAMP antagonists e.g. a thiosubstituted cAMP analog (such as derivatives of adenosine-3 ', 5 ' -cyclic monophosphorothioate, Rp isomer, such as Rp-8-Br-cAMPS or Rp-8-Cl-cAMPS) , or COX-2 inhibitors as described in WO02/07721, which is incorporated herein by reference. Other examples, e.g. in the treatment of HIV or AIDS preferably the inhibitor is used in combination with one or more NNRTIs (non-nucleoside reverse transcriptase inhibitors) or in combination with one or more NRTIs (nucleoside reverse transcriptase inhibitors) or in combination with one or more HIV protease inhibitors or one or more HAART (highly active antiretroviral therapy) in combination with the inhibitor of this invention. Thus, pharmaceutical compositions of the invention may additionally contain one or more of such active ingredients . In a further aspect, the present invention provides methods and/or compositions which combine one or more inhibitors as described herein with compounds that improve the tolerability of the active ingredient, especially during long term treatment. Typical compounds include antihistamine and proton pump inhibitors . According to a yet further aspect of the invention we provide products containing one or more inhibitors as herein defined and one or more additional active ingredients as a combined preparation for simultaneous, separate or sequential use in human or animal therapy.
The following Examples are given by way of illustration only in which the Figures referred to are as follows:
Figure 1 shows the identification of AKAPs in T cell lipid rafts in which lysed Jurkat T cells fractionated on sucrose gradients were resolved on SDS-PAGE, blotted to PVDF-membranes, subjected to Rll-overlay assay (A) and probed with the indicated antibodies (B) . Immunodetection of LAT-antibody was used as a marker for lipid rafts. Lanes refer to fraction numbers following sucrose gradient centrifugation of the lysate and fractionation from the top. Mobility of molecular weight markers are indicated;
Figure 2 shows deletional mapping of the PKA anchoring domain in ezrin. Schematic view of ezrin fragments expressed and tested for interaction (A, left) . Depicted are the N-terminal (dark shaded) ; oi-helical (light shaded) and C-terminal (black) domains of Ezrin. Fragments were tested for interaction with Rio. and RIIo; in the yeast-2 -hybrid system by growing on selective media and examining β-gal (A, middle panel) . GST-linked ezrin fragments as indicated were incubated separately with Rio; and RIIo. proteins and precipitated using glutathione-agarose beads (A, right) . GST-pulldown with GST without fusion in presence of PKA subunits served as negative control. Yeast lysates expressing VP16-HA Rlα- subunit and lexA-ezrin constructs (B, two upper panels) were immunoprecipitated with an i-HA antibody and precipitates were subjected to anti-lexA immunoblotting to identify interactions (B, bottom panel) ;
Figure 3 shows the determination of association and dissociation rate constants for the binding of Ezrin to the Rio;-subunit .
An Ezrin clone isolated by yeast-2-hybrid assay (amino acids 280-585) using Rlα-bait was inserted into a GST- expression vector in the sense (+) and anti-sense (-) orientation and transformed into E. Coli (BL21) . GST- fused proteins were purified after incubation in the absence or presence of IPTG at 0 and 3 h and subjected to SDS-PAGE, blotted to PVDF membrane and incubated with recombinant human Rio; protein (100 nM) or radiolabelled RIIo; (5 nM) washed and immunoblotted with anti-Rio. antibody or directly subjected to autoradiography (A) . Interaction studies were performed with a fixed amount of immobilized Rio; subunit (B) . Ezrin (the fragment 280-470) at concentrations indicated on the plot were injected for 300 s and after the end of the injection the dissociation phase without any AKAP present was monitored for 300 s (B) . Qualitative surface competition experiments with Ht31 peptide (amino acids 493-515) (C) . Rio., was immobilized as above and ezrin (fragment, as above) in a concentration of 500 nM was injected in the absence (middle trace) or presence of Ht31 competitor peptide (10 mM; Ht31, lowest trace) . As a control, 10 mM of a peptide containing a proline residue in the AKAP interaction site (Ht31P, upper trace) was injected with ezrin;
Figure 4 shows the effect of actin disruption by cytochalasin D on distribution of AKAPs in lipid rafts.
Lipid rafts were purified from T lymphocytes by sucrose gradient centrifugation following incubation in the absence or presence of cytochalasin D (80 mM) . The same experiment without cytochalasin treatment was performed in parallel. Fractions were resolved on SDS-PAGE, blotted to PVDF-membranes and probed with the indicated antibodies (A, B two different fractions) . Lanes as indicated in Figure 1. Immunoblotting with anti-LAT antibody indicated lipid raft containing fractions. Molecular weight markers are indicated;
Figure 5 shows that ezrin interacts with PKA type Io. in T-cell lipid rafts.
Lipid raft fractions of T lymphocytes were isolated, pooled and subjected to precipitation with mouse monoclonal antibodies against Rio, RIIo or control IgG (A) . Precipitates were analyzed by 10% SDS-PAGE followed by immunoblotting with mouse monoclonal antibodies against Ezrin (upper panel) , RIo;, RIIoc and PKA-C subunits (lower panels) . Similarly, lipid raft fractions were subjected to immunoprecipitation with anti-ezrin antibodies, precipitated and immunoblotted with the indicated antibodies (B) ;
Figure 6 shows that ezrin colocalizes with the RIo.- subunit of PKA in T cell lipid rafts .
Jurkat-T cells were immunostained using (A) a mouse monoclonal antibody against RIo; and (B) rabbit polyclonal antibody against the lipid raft specific protein LAT; rabbit polyclonal antibody against ezrin (D,G) and a mouse monoclonal antibody against RIo; (E) or RIIo (H) . Merged images show overlapping subcellular distribution (C,F,I);
Figure 7 shows that the RIo;-subunit of PKA is distributed with VSVG-tagged ezrin.
Jurkat T-lymphocytes transfected with cDNA constructs coding for VSVG-tagged (full-length) ezrin were stained with mouse monoclonal antibody VSVG (A) and mouse monoclonal IgG2A antibody against RIo. (B) . Colocalization of RIo; with Ezrin-VSVG appears as signal in the merged image (C) . Control immunocytochemical analysis showed no antibody signal. Scale bar, 5 μm;
Figure 8 shows that ezrin is colocalized with PKA, EBP50 and Csk in T-cell lipid rafts.
Lipid raft fractions of T lymphocytes were isolated, pooled and subjected to precipitation with rabbit polyclonal against Csk or control IgG (A) . Precipitates were analyzed by 10% SDS-PAGE followed by immunoblotting with mouse monoclonal antibodies against ezrin (upper panel) or EBP50 (lower panel) . Similarly, lipid raft fractions were subjected to immunoprecipitation with mouse monoclonal anti-EBP50 antibodies, precipitated and immunoblotted with antibodies to Csk or PKA-C (B) . Figure 8C is a schematic of the complex formed during PKA type I signalling;
Figure 9 shows the expression of soluble R-binding domains of ezrin and competition of Ht31 to remove anchored RIx from lipid rafts and disrupt cAMP inhibition of T cell function.
T lymphocytes were transfected with mammalian expression vectors encoding PKA-binding domains of ezrin, or empty vector and incubated for 16 h. Subsequently, cells were incubated with or without 50 μM (15 min) cAMP and next stimulated with anti-CD3£ mAb OKT3 and incubated for 20 h. Supernatants were harvested and IL-2 levels were determined by ELISA and the effect of anchoring disruption on cAMP-mediated inhibition of IL-2 production assessed (A) . The release from inhibition by cAMP was also calculated as fold increase in IL-2 production upon disruption of PKA anchoring (B) ;
Figure 10 shows the identification of a putative upstream type I regulatory subunit binding domain (RI- AKAP specifier region) in PKA type I binding AKAPs using bioinformatics . The alignment was performed using the Clustal-W program. The names of the AKAPs and residue numbers of the binding domains are indicated on the right. Boxed text shows conserved residues with similar functional properties. In addition, the down-stream amphipathic helix binding domain that may confer binding to both PKA types I and II (albeit with different specificities) is shown. In the alignment, the locations of additional residues with structural similarity are marked by stars,-
Figure 11 shows analysis of binding of PKA type I and II to various parts of ezrin by peptide array. Scanning arrays of the ezrin polypeptide were synthesized (Autospot, Intavis AG) where each peptide (spot) represents a 20-mer with a 2-residue frame shift from the previous peptide. The immobilized peptides were analyzed for R binding by either RII-32P-overlay with wild type RIIo. or by overlay with RI-32P RIo.(A98S) or 1-11, 1-15 or 1-24 N-terminal deletions of RIo;(A98S) as indicated above the columns. Binding of 32P-labeled RI or RII was detected by autoradiography. Regions that appear to bind in the scan analysis are shown;
Figure 12 shows lowered sensitivity to cAMP in inhibition of immune responses in mature T cells with Ezrin knockdown. (B) Purified T cells were transfected with mutated control si RNA (mock) or Ezrin-specific (Ez799) siRNAs. 48 hours later cells were pretreated with 8-CPT-cAMP (0, 10 or 50 μM) and either kept unstimulated or stimulated for 20 hours with anti-CD3/anti-CD28 coated beads (bead-to-cell ratio 1:1) . Then supernatants were harvested and assessed for IL-2. Levels of IL-2 secretion are shown relative to those of anti-CD3/anti-CD28 stimulated cells (average ± S.E.M., n=6-9) . (D) As in B, but with different ezrin-specific (Ez2145) and control (mock) siRNAs and duplicate measurements (average ± half range) . Immunoblots verifying Ezrin knockdown are also shown (A and C) .
EXAMPLE 1 : Identification of Ezrin as an anchoring protein for PKA type I and EBP50
Materials and Methods
Yeast-Strains and Media
Saccharomyces cerevisiae yeast strains were grown at 30°C in standard liquid YPD medium or minimal SD synthetic medium with appropriate supplement amino acids (Clontech Laboratories, Inc.) .
Yeast- two -hybrid screening
Screening of a normal lymphocyte library was performed using the Matchmaker two-hybrid system (Clontech Laboratories, Inc.) . The full-length Rlα subunit of PKA was subcloned into pAS2.1 and cotransformed together with the cDNA-library (Clontech catalog no. HL4014AB) into Y90 yeast cells. HIS-positive clones were further selected by colony lift filter assay for β-galactosidase activity. Plasmid DNA was reduced in E. coli DH5 o. from yeast cells. Plasmids were retrans ormed into yeast reporter strain HF7c (genotype: MATa, ura3-52, his3-200, lys2-801, ade2-101, trpl-901, leu2-3, 112, gal4-542, gal80-538, LYS : :GAL-HIS3 , URA3 : : (GAL4 17mers)3- CYCl-lacZ from Clontech) with plasmid pGBT-PBR to test for histidine prototrophy and β-galactosidase activity (Clontech, manual) . The cDNA inserts from the positive clones were sequenced.
Yeast two -hybrid analysis
Ezrin constructs were cloned into the EG202 bait vector and RIo; and RIIo; into the JG4-5 prey vector. Mating assays and detection of interactions were performed as described (Gronholm et al . , 1999, J. Cell Sci., 112, p895-904) . Cell Cul ture, Stimulation, and Transfection of Jurkat TAg
The human leukemic T cell line Jurkat TAg, a derivative of the Jurkat cell line was kept in logarithmic growth in RPMI medium supplemented with 10% fetal calf serum and antibiotics. For transfections, T-cells (2 x IO7) in 0.4 ml of Opti-MEM were mixed with 20 μg of Ht31-cDNA construct in electroporation cuvettes with a 0.4 cm electrode gap (Bio-Rad) and subjected to an electric field of 250 V/cm with 960-microfarad capacitance . The cells were expanded in complete medium and harvested after 20 h.
Negative selection of peripheral blood CD3+ T cells
Peripheral blood CD3+ T cells were purified by negative selection from buffycoats from normal healthy donors (Ullevaal University Hospital Blood Center, Oslo, Norway) . Briefly, peripheral blood mononuclear cells were isolated by density gradient (Lymphoprep, NycoMed, Oslo, Norway) centrifugation followed by negative selection using monodisperse magnetic beads directly coated with antibodies to CD14 and CD19 and rat anti- mouse IgG beads coated with antibodies to CD56 and a magnet. Magnetic beads were all from Dynal (Oslo, Norway, cat. no. 111.12, 111.04 and 110.11, respectively) whereas anti-CD56 antibody was from Pharmingen (San Diego, CA, cat. no. 31660. d) . All steps were performed at 4°C. Cell suspensions were analyzed by flow cytometry and routinely shown to consist of more than 90% CD3+ cells.
Transf ections of T-cells
For transfections, T-cells (5xl06) in 0.1 ml of Nucleofection solution (Amaxa) were mixed with 2 μg of each cDNA construct and subjected to electroporation in a Nucleofector (Amaxa) following the manufacturer's protocol. The cells were expanded in complete medium and incubated for 16h at 37°C. 4x 10s T-cells were treated with 50 μM cAMP for 15 min (37°C) and activated by the addition of 5 g/ml anti-CD3 ε mAb OKT-3. After 16 h incubation at 37°C the level of secreted IL-2 was determined by ELISA.
Fractionation of detergent- resistant GEMs (lipid rafts) by densi ty gradient ul tracentrifugation
Primary T-cells or Jurkat T-Cells (3x10s cells obtained from confluent culture flasks) were washed in PBS, resuspended in 1ml of MES containing 80 μg/ml Cytochalasin D and pelleted after 30 min at 37°C. T- cells were resuspended in 1 ml ice-cold lysis buffer (25 mM MES, pH 6.5, 100 mM NaCl, 5 mM EDTA, 0,7% Triton X-100 with 1 mM sodium orthovanadate, 1 mM PMSF, 10 mM sodium pyrophosphate, and 50 mM sodium fluoride) containing 0.7% Triton X100 and lysed 10 min on ice. After 10 strokes with dounce-glass-homogenizer, the lysate was mixed 1:1 with 80% (w/v) sucrose and placed at the bottom of a 5.2 ml polyallomer centrifuge tube (Beckman Instruments), then carefully overlaid with 2.0 ml of 30% (w/v) sucrose in MNE-buffer and finally with 1 ml of 5% (w/v) sucrose in MNE-buffer.
Centrifugation was performed at 4°C in a Beckman SW55Ti rotor (20 h, 46000 rpm) . Twelve 0.4 ml fractions were collected gradually from the top of the gradient, proteins were separated by SDS-PAGE and analyzed by immunoblotting.
Immunoprecipi tation and Western Blotting
1-5 μg antibody and 25 μl of protein A/G plus agarose (SC-2003, Santa Cruz Biotechnology) were added to T-cell lipid raft fractions. Precipitates were washed three to four times with lysis buffer containing 0.1-1% Triton X100, resolved by 10% SDS-PAGE under reducing conditions, and transferred by semidry electroblotting to polyvinylidene difluoride membranes . Blots were blocked with 5% dry milk in PBS-T (PBS/ 0.1% Tween 20), before incubation with the indicated Ab. After extensive washings with PBS-T, blots were incubated with either horseradish peroxidase HRP-conjugated goat anti-mouse or goat anti-rabbit IgG, washed, and developed with enhanced chemilu inescence (Supersignal Pierce) .
RI/RII overlay
Ezrin fragments fused to GST were produced in Escherichia coli BL21 cells following stimulation with IPTG (3h) . BL21 cells were pelleted from 10 ml of broth, lysed and after removal of insoluble pellet, glutathione beads were added for lh at 4QC. The beads were then washed and finally boiled in SDS-PAGE buffer. Proteins were separated on 10% polyacrylamide gel . For RI overlay, membranes were blocked in "blotto" overnight at 4°C, 100 nM GST-cleaved RIo: was added to "blotto" and incubated with the membrane for 4-6 h at 4°C. Membranes were then washed with PBS and blocked for Western blot analysis against RIo;.
RIIo; overlay was performed as previously described (Carr & Scott, 1992, Trends Biochem. Sci., 17, p246-249). Briefly, bovine RIIo; was radiolabelled with [γ-3ZP] -ATP and unincorporated ATP was removed by gel filtration. Labelled RIIo was then incubated with the membrane overnight and washed extensively to remove non-specific binding. GST-precipitation assay
Fragments of ezrin fused to GST were expressed in Escherichia coli BL21 cells, induced using 0.4 mM IPTG and purified on glutathione-sepharose (Amersham Pharmacia Biotech) . 2.5 μg of purified ezrin fragments and R subunits were incubated at a 1:1 ratio (50 μM each) in 100 μl pull-down buffer (300 mM NaCl, 0.1 % Triton X-100, lmM PMSF, lmM EDTA, 5mM benzamidine, 5 mM DTT, lOμg/ml of antipain, chymostain, leupeptin and pepstatin A) overnight at 4°C. Glutathione-sepharose beads equilibrated in pull-down buffer with 1% BSA were added and incubated with rotation for 30 minutes at 4°C. The beads were washed with pull-down buffer five times for 5 min and boiled in SDS-PAGE buffer. Proteins were resolved on an 8-16% SDS-polyacrylamide gel and analysed by immunoblotting for the presence of R subunits. Monoclonal antibodies directed against human RIo; and human RIIo; (cat. nos . P53620 and P55120, respectively; KT in collaboration with Transduction laboratories, Lexington, KY) were used at a concentration of 1.0 μg/ml .
Peptide loading of peripheral T cells
In order to facilitate uptake of peptides into cells we used liposome mediated transfection. Peptide at 25- lOOnM, dissolved in lOmM Hepes pH 7.4, was mixed with 3 μl of liposomes (DOTAP, Boehringer Mannheim) and brought to a total volume of 10 μl before being added to the cells (75 000 in 90 μl) . The optimal concentrations of DOTAP and anti-CD3 antibody were titrated carefully in order to maintain membrane stability and normal TCR/CD3- dependent activation of the T cells . Cells were incubated for 20 hours with DOTAP/peptide after which cells were cultured in the absence or presence of 8-CPT- cAMP (6.25 μM) and induced to proliferation via TCR/CD3 activation. Results
Cloning of T cell AKAPs for PKA types I and II
In order to first pursue an open strategy to identify T cell AKAPs, we screened separately for the presence of AKAPs interacting with PKA types I and II. To look for RI binding proteins in T cells, we used the yeast two- hybrid system, which allows for in-solution interaction rather than filter overlay interaction (Huang et al . , 1997, PNAS, 94, plll84-11189 ; Huang et al . , 1997, J. Biol. Chem., 272, p8057-8064) . For the identification of RII binding proteins the well-established Rll-overlay screening was used (Carr & Scott, 1992, supra,- Witczak et al , 1999, EMBO J., 18, pl858-1868 ; Schillace et al , 2002, J. Immunol., 168, pl590-1599) . Screening of a human lymphocyte yeast two-hybrid library using the full-length RIo; subunit as a bait and an RIαΔl-45 deletion mutant lacking the dimerization- and AKAP- binding-domains as control (Carlson et al . , 2003, J. Mol. Biol., 327, p609-618) identified 12 clones that interacted with the full-length but not the deleted RIo; bait. Of the 12 clones, 2 were identified as ezrin, 2 were identified as PAP7 , a itochondrial AKAP for PKA type I as earlier reported (Li et al . , 2001, Mol. Endocrinol . , 15, p2211-2228) and the remaining 8 were checked out as false positives.
To account for the presence of AKAPs for PKA type II in T cells, an RII overlay screening of a Jurkat T cell Lambda Zap expression library was performed using radio- labelled RIIo; as a probe. In contrast to the two-hybrid library screening with RIo; as a bait, this screening identified AKAPs 95, 79, 149 and a fragment of AKAP450 in T cells, which is consistent with the recent report by Carr and co-workers on PKA type II AKAPs in T cells (Schillace et al . , 2002, supra). Notably, the screening with RIo; as a bait identified a different set of AKAPs in T cells to those identified by the RII overlay screening.
Identification of A kinase anchor proteins associated wi th T cell lipid rafts
To identify AKAPs that specifically would serve to anchor the pool of PKA type I identified in T cell lipid rafts and that are involved in the PKA type I- Csk inhibitory pathway that regulates TCR signalling, we performed sucrose gradient fractionation of Jurkat T cell lysates and performed far-Western experiments (RII overlay) using radio-labelled PKA RIIo; subunit. Fig. 1A shows the result of this experiment, which identified R- binding proteins of approximately 80 and 150 kDa.
Parallel sucrose gradient fractionations of lipid rafts were next subjected to immunoblot analysis with antibodies to known AKAPs of the approximate molecular weights of the AKAPs identified in Fig. 1A. The 80-kDa R-binding protein which was abundant in lipid rafts compared to non-raft fractions (compare fractions 3 to 5 versus fractions 9 to 12) , was identified as ezrin which has a size of 78 kDa (Fig. IB) . In contrast, AKAP79 which is of approximately the same molecular weight was not detected in lipid rafts. Furthermore, the same lipid raft fractions, identified by immunoblot analysis with the lipid raft marker LAT, were also shown to contain PKA Rlα and C subunits (Fig. IB) as earlier reported (Vang, 2001, supra) .
The approximately 150-kDa band detected in Fig. 1A was further identified as AKAP149 (D-AKAP1) . However, the following observations made us focus on the functional role of ezrin as a lipid raft anchoring protein for PKA in T cells: i) Ezrin was identified both in a yeast two- hybrid screen and as an R-binding protein purified from lipid rafts; ii) AKAP149 was less abundant in lipid rafts versus non-raft fractions (compare fractions 4 to 5 and 9 to 12) ; and iii) Disruption of PKA-AKAP149 signalling complexes did not appear to affect signalling through the PKA-Csk-Lck regulatory pathway in T cell lipid rafts (not shown) .
Mapping and characterization of the R-binding domain of ezrin
We next used the yeast two-hybrid system for a deletional mapping analysis of determinants for interactions with RIo; and RIIo; as assessed by growth on drop-out media and β-galactosidase activity. This showed that the smallest fragment of ezrin interacting with PKA comprises amino acids 363 to 470 that covers the α-helical region of ezrin (Fig. 2A, left and middle) . Notably, the interaction appeared stronger with RIo; than with Rllα. Further deletional mapping to a smaller fragment (amino acids 404 to 445) containing an amphipathic helix earlier predicted as a putative R- binding domain (Dransfield et al . , 1997, EMBO J., 16, p35-43) , did not show binding in the two-hybrid system but still showed some weak binding versus RIo; using GST- ezrin in an in vi tro precipitation assay (Fig. 2A, right columns) . Furthermore, both the results from the GST- ezrin precipitations (Fig 2A) and immunoprecipitation of tagged proteins from yeast cell lysates with the bait and pray proteins (Fig. 2B) verified the observations made in the yeast two-hybrid system. However, full- length ezrin containing both the globular and N-terminal domains did not bind PKA. This may be due to the fact that the unbound, inactive ezrin assumes a folded conformation where the FERM (four-point-one, ERM) domain in the N-terminus binds the globular domain (g-ezrin) (Gary R et al . , 1995, Mol. Biol. Cell, 6(8), pl061- 1075) . In this state the PKA binding site may be inaccessible by steric hindrance.
We next examined in more detail the binding of PKA to the fragment of ezrin (amino acids 280 to 470) identified in the two-hybrid screenings. Using a filter overlay assay we showed that both RIo and RIIo; bound to a GST-fusion protein resulting from expression of the clone in bacteria in the sense orientation, but not in the antisense orientation (Figure 3A) .
We subsequently purified the same fragment and examined the binding of ezrin to the RIo; subunit of PKA immobilized on a cAMP chip by surface plasmon resonance (Fig. 3B) . Interaction was monitored in 20 mM MOPS, 150mM KC1, lmM DTT and 0.005% surfactant P 20 at a flow rate of 30 μL/min. After subtracting a blank surface, a Langmuir 1:1 model for the interaction was assumed using a global fit analysis algorithm provided in Biaevaluation 3.0. This analysis, using a range of concentrations of ezrin fragment indicated a nanomolar affinity (KD 25 nM) for the interaction of ezrin with Rlα, whereas similar experiments with RIIo; demonstrated an approximately 10-fold higher affinity (KD 1.4 nM; not shown) .
Furthermore, binding of both RIo; (Fig. 3C) and RIIo; (not shown) were fully displaced with 10 nM of the Ht31 competitor peptide whereas no effect was seen with the Ht31-P control peptide. Since Ht31 binds to the immobilized R-subunits, a small increase in mass was expected. Therefore control experiments were performed for all interactions using 10 nM of Ht31 (Ht31) and Ht31p (Ht31P) without AKAP present. PKA - ezrin complexes are present in lipid rafts and attached to the cytoskeleton
Having shown that f-ezrin (fibrous ezrin) but not g- ezrin (globular ezrin) binds PKA in vi tro (Figures 2A and 2B) , the possibility existed that PKA would mainly be associated with active ezrin that was not only anchored to lipid raft membrane proteins such as CD44, but also to the actin cortical cytoskeleton in T cells.
Cytoskeletal elements separate to dense fractions in the gradients used to purify rafts and ezrin-actin interactions may persist during the fractionation and effectively serve to rip ezrin-PKA complexes out of rafts. In order to optimize separation of the lipid raft-associated pool of ezrin and examine association with PKA, we prepared lipid rafts from T cells in the presence and absence of the actin-depolymerizing drug cytochalasin D. When actin cytoskeleton was disrupted, we observed increased amounts of ezrin in lipid rafts (Fig. 4A).. Also PKA RIo. and some levels of PKA RIIcc were present in the same raft preparations (Fig. 4A) . Notably, the relative amount of Rlα in raft fractions, as opposed to non-raft fractions, increased after cytochalasin D treatment. The ezrin-binding protein EBP50 also co-migrated with ezrin in the density gradient separation and localised to lipid rafts (Fig. 4B) .
We next performed immunoprecipitations from cytochalasin D treated preparations of T cell lipid rafts. Results from these experiments showed that immunoprecipitations using RIo; or RIIo; antibodies co-precipitated both ezrin and the PKA C subunit (Fig. 5A) . Conversely, immunoprecipitation with ezrin antibodies co- precipitated RIo and C as well as smaller amounts of RIIo; (Fig. 5B) . This indicates that although ezrin is able to bind both RIo; and RIIo., the major pool of PKA associated with ezrin in T cell lipid rafts is a PKA type I (RIo-2 C2) .
Immunofluorescence studies on the localization of PKA. type I and ezrin showed that the majority of PKA type I (RIo;) co-localized with the lipid raft marker LAT (Fig. 6A-C) . Furthermore, dual immunostaining for ezrin and RIo; again demonstrated that a major pool of ezrin and PKA type I (RIo;) appear to be co-localized in the proximity of the plasma membrane as evident from the co- localized and patched staining for RIo; and ezrin (Fig. 6D-F) . In contrast, double immunofluorescence staining of RIIo. and ezrin demonstrated that a majority of RIIo. localized to the Golgi-centrosomal region in T cells which contained no ezrin (Fig 6G-I) . Immunostaining for ezrin and RIo; on isolated mononuclear peripheral blood cells verified that PKA type I (RIo;) and ezrin are co- localized also in normal cells (Fig 6J-L) .
We next examined the distribution of ezrin and RIo. after over-expression of a VSVG-tagged ezrin in Jurkat T cells by double immunofluorescence staining. RIo. co-localized well with the expressed ezrin (Figs. 7A-C) . Furthermore, overexpression of ezrin resulted in numerous needle-shaped processes at the cell surface (philopodia) as reported elsewhere earlier. Notably, RIo; also localized to these needle-shaped ezrin containing processes at the cell surface.
EBP50 bridges ezrin and Cbp/PAG to yield a scaffold for the PKA-Csk signalling pathway in T cell lipid rafts
Since previous observations indicate that both ezrin and Cbp/PAG can be bound to the linker protein EBP50, we examined the possibility that EBP50 cross-links the PKA and Csk anchoring proteins by simultaneous contact with both. We earlier reported that the lipid raft pool of Csk is exclusively localized to Cbp/PAG (Vang et al . , 2001, supra; Vang et al . , 2003, J. Biol. Che . , 278, pl7597-17600) . Immunoprecipitation of the Csk-Cbp/PAG complex by anti-Csk antibodies revealed that EBP50 (as earlier reported, (Brdickova, 2001, supra) as well as ezrin were present in the Csk immunoprecipitates (Fig. 8A) . Furthermore, both Csk and the PKA C subunit were detected by immunoblot analysis of EBP50 immunoprecipitates (Fig. 8B) . This indicates the presence of a PKA/ezrin/EBP-Cbp/PAG/Csk signalling complex positioning PKA in close proximity to Csk for its phosphorylation and regulation of Csk activity (shown schematically in Fig. 8C) .
EXAMPLE 2 : Inhibition of PKA Type I signalling by disruption of ezrin binding
Results
Disruption of PKA type I anchoring in lipid rafts reverses the inhibi tory effect of cAMP on T cell immune functions
In order to abolish the effect of anchored PKA type I in T cell lipid rafts, we attempted to compete its localization using soluble peptides or expressed AKAP fragments containing the PKA binding domain. The Ht31 anchoring disruptor containing the R-binding site from AKAP-Lbc is well established and widely used to compete localization of PKA. Although Ht31 has a higher affinity for PKA type II, it can also be used to disrupt PKA type I interaction with AKAPs (Fig. 3C, Herberg et al . , 2000, J. Biol. Chem., 298, p329-339) . Indeed, when T cells were transfected with a construct directing the expression of the Ht31 fragment (Lester et al . , 1997, PNAS, 94, pl4942-14947) this displaced both PKA types I and II from lipid rafts (not shown) .
Peripheral blood T cells were transfected by nucleofection (Amaxa) with constructs directing the expression of a soluble ezrin fragment containing the cx-helical domain mock-transfected. Transfected and control-transfected cells were either left untreated, activated by cross ligation of the TCR/CD3 complex or pretreated with a concentration of cAMP that produced an 80% maximal inhibition of IL-2 production, and subsequently activated and cultured for 16 hours after which IL-2 levels secreted to the supernatant were assessed. Results from these experiments show that T cells expressing soluble fragments of ezrin were less sensitive to cAMP-mediated inhibition of IL-2 production (Fig 9A) . IL-2 production in the presence of cAMP increased 3-fold by introducing soluble fragments of ezrin (Fig. 9B) .
EXAMPLE 3 : Identification of a putative upstream type I regulatory subunit binding domain (RI-AKAP specifier region) in AKAPs
Using the Clustal-W program a putative upstream type I regulatory subunit binding domain (RI-AKAP specifier region) was identified in PKA type I binding AKAPs . The results are shown in Figure 10. A conserved sequence in the region of 367-390 (numbering according to ezrin) was identified. EXAMPLE 4 : Protein kinase A type I , but not protein kinase A type II. binds to the upstream RI-AKAP specifier region in ezrin
Methods
Auto spot Peptide Array
Peptide arrays were synthesized on cellulose paper by using an Autospot Robot ASP222 (ABiMED, Langenfeld, Germany) as described (Frank, R. , 1992, Tetrahedron 48, 123-132) .
R- overlay
R-overlays were conducted as described, using 3ZP-labeled recombinant murine RIIo; (Hausken, Z. E. , et al . , 1998 in Protein Targeting Protocols, ed. Clegg, R. A. (Humana, Totowa, NJ) , Vol. 88, pp. 47-64), recombinant bovine RI (A98S) or deletion mutants.
Results
Using radiolabeled RI as a probe, binding to regions 356-375 and 378-397 was detected. This overlapped with the predicted upstream RI-AKAP specifier region in ezrin. Furthermore, this region appeared to have a tandem binding motif. In contrast, no binding to this region was detected with radiolabeled RII or with any of the RI deletion mutants indicating that the RI-AKAP specifier region interacts with the N-terminus of RIo.
In addition, binding was detected to peptides with amino acids 414 to 443 containing the predicted amphipathic helix region of ezrin known to bind PKA type II (amino acids 414 to 430 indicated in italics in Fig. 11) . RII-32P overlay demonstrated binding to the most N-terminal part of this region whereas RI-32P overlay gave weak binding to the more C-terminal part of the helix. The RI-binding was enhanced when the N-terminus had been removed.
EXAMPLE 5: siRNA-mediated knockdown of Ezrin disrupts cAMP inhibition of T cell proliferation
Methods
21-nt siRNA duplexes targeting different positions within human ezrin mRNA were designed and synthesized in-house. Following initial screening of siRNA activity in cell culture, mismatched control siRNAs for the best siRNA were also designed. SiRNAs are named according to the position of the 5 ' nucleotide of the sense strand relative to the reference sequences of the respective target mRNAs and the sequences are:
Ezrin 799 si RNA: 5'- cccuuggacugaauauuuaug -3' and 5'- uaaauauucaguccaagggca -3 ' ,-
Ezrin 2145 si RNA: 5'- gga gca gga cug auu gaa uua -3' and 5'- auu caa ucu guc cug cue cca -3'.
Primary T cells from healthy Norwegian blood donors were purified by negative selection (Aandahl EM et al . , 1998, FASEB J. 12:855-62) and transfected with siRNA (50-400 nM) in accordance with the manufacturers ' instructions using the Amaxa nucleofector and kit (cat. no. VPA-1002) . Cells were then incubated for 48 hours in complete medium before harvesting.
Primary T cells 48 hours post-transfection were stimulated with anti-CD3/antiCD28 coated beads (Dynal, cat no. 111.31, different numbers of beads per cell) for 20 hours, thereafter supernatants were harvested and the concentration of IL-2 was assessed by ELISA (R&D Systems, cat co. D2050) . As control, transfected cells were stimulated for 20 hours with PMA/Ionomycin (40nM and lOμM, respectively) .
Results
Knock down of Ezrin protein levels was achieved after 24 to 48 h of transfection to an efficiency of approx 80% or more in peripheral T cells in different experiments. When the sensitivity of anti-CD3/anti-CD28 -induced IL-2 secretion to increasing doses of cAMP was assessed, it was clear that in cells where ezrin had been knocked down the IL2-responsiveness in the presence of cAMP increased illustrating inactivation of the PKA type I signalling pathway. Furthermore, this information provides evidence that ezrin is an integral part of the complex PKA type I / ezrin / EBP-50 /Cbp/PAG /Csk and that ezrin is required for modulation of functional immune responses by cAMP.

Claims

Claims :
1. A method of altering the PKA type I signalling pathway in a cell comprising the step of administering an inhibitor, or a molecule encoding an inhibitor, which reduces or inhibits the binding between one or more of the following binding partners : i) ezrin and PKA type I, ii) ezrin and EBP50, and iii) EBP50 and Cbp/PAG.
2. The method of claim 1 wherein the PKA type I signalling pathway is down-regulated.
3. The method of claim 1 or 2 wherein said cell is a lymphocyte, preferably a T lymphocyte. . The method of any one of claims 1 to 3 wherein said inhibitor is selective for PKA type I relative to PKA type II signalling.
5. The method of any one of claims 1 to 4 wherein said inhibitor is selective for PKA type Iα relative to PKA type Iβ signalling.
6. The method of any one of claims 1 to 5 wherein said binding partners ezrin, PKAI, EBP50 and Cbp/PAG are polypeptides comprising SEQ ID NO. 1, 2, 3 and 4, respectively, or their functionally equivalent variants having more than 75% sequence similarly thereto, or derivatives or fragments thereof .
7. A method of altering the PKA type I signalling pathway in a cell comprising the step of administering an inhibitor, or a molecule encoding an inhibitor, which reduces or inhibits the binding between one or more of the following binding partners: i) a polypeptide comprising the sequence as set forth in SEQ ID No. 1 or a sequence with 95% similarity thereto or a sequence encoded by a nucleotide sequence which hybridizes under conditions of high stringency to the nucleotide sequence encoding the amino acid sequence of SEQ ID No. 1, or a functionally equivalent fragment thereof, and a polypeptide comprising the sequence as set forth in SEQ ID No. 2 or a sequence with 95% similarity thereto or a sequence encoded by a nucleotide sequence which hybridizes under conditions of high stringency to the nucleotide sequence encoding the amino acid sequence of SEQ ID No . 2, or a functionally equivalent fragment thereof; ii) a polypeptide comprising the sequence as set forth in SEQ ID No. 1 or a sequence with 95% similarity thereto or a sequence encoded by a nucleotide sequence which hybridizes under conditions of high stringency to the nucleotide sequence encoding the amino acid sequence of SEQ ID No . 1, or a functionally equivalent fragment thereof, and a polypeptide comprising the sequence as set forth in SEQ ID No. 3 or a sequence with 95% similarity thereto or a sequence encoded by a nucleotide sequence which hybridizes under conditions of high stringency to the nucleotide sequence encoding the amino acid sequence of SEQ ID No. 3, or a functionally equivalent fragment thereof ; iii) a polypeptide comprising the sequence as set forth in SEQ ID No. 3 or a sequence with 95% similarity thereto or a sequence encoded by a nucleotide sequence which hybridizes under conditions of high stringency to the nucleotide sequence encoding the amino acid sequence of SEQ ID No. 3, or a functionally equivalent fragment thereof, and a polypeptide comprising the sequence as set forth in SEQ ID No. 4 or a sequence with 95% similarity thereto or a sequence encoded by a nucleotide sequence which hybridizes under conditions of high stringency to the nucleotide sequence encoding the amino acid sequence of SEQ ID No . 4, or a functionally equivalent fragment thereof .
8. The method of any one of claims 1 to 7 wherein said binding partners comprise fragments of said polypeptides which correspond to the binding sites between said binding partners .
9. The method of claim 8 wherein said binding partner polypeptides comprise the following amino acid sequence or a sequence with 95% similarity thereto or a sequence encoded by a nucleotide sequence which hybridizes under conditions of high stringency thereto: (a) amino acids 356-470 or 1 to 286 of SEQ ID No. 1; or (b) amino acids 1 to 50 of SEQ ID No. 2; or (c) amino acids 11-97, 149-236 or 329-358 of SEQ ID No. 3 ; or (d) amino acids 429 to 432 of SEQ ID No . 4.
10. The method of any one of claims 1 to 9 wherein said inhibitor directly interferes with the binding at the binding site of one or more of the binding partners as defined in any one of claims 1 or 6 to 9.
11. The method of any one of claims 1 to 10 wherein said inhibitor specifically recognises the binding site of one or more of the binding partners as defined in any one of claims 1 or 6 to 9.
12. The method of any one of claims 1 to 11 wherein said inhibitor is an antibody or antigen binding fragment thereof, a peptide, a protein, or a nucleic acid molecule which encodes said antibody, peptide or protein.
13. The method of any one of claims 1 to 11 wherein expression of the endogenous gene encoding a binding partner as defined in any one of claims 1 or 6 to 9 is manipulated, wherein preferably said inhibitor is a sense nucleotide sequence, an antisense nucleotide molecule, ribozyme or RNAi .
14. The method of any one of claims 1 to 13 wherein said inhibitor is a modified endogenous peptide, or nucleic acid encoding said peptide, wherein the unmodified version of said endogenous peptide is a binding partner as defined in any one of claims 1 or 6 to 9, and said peptide is modified by one or more mutations in one or more binding sites of said binding partner, in which said mutation results in a molecule that has impaired binding at that site to its binding partner.
15. The method of claim 14, wherein said inhibitor is a modified peptide from ezrin, in which one or more of the valine and/or isoleucine residues in the PKA binding site of the ezrin molecule, preferably residues 363 to 470 of SEQ ID No. 1, is mutated, preferably to a proline residue .
16. The method of any one of claims 1 to 15 wherein said inhibitor reduces or inhibits binding between ezrin and PKA type I and wherein said inhibitor mimics the PKA I binding site on ezrin, preferably mimics residues 356 to 470, preferably residues 356-397, 363-390, 397-470, 390-470, 356-376, 363-404, 363-412, 367-390, 378-397, 390-412, 413-420, 413-430, 414-443 or 421-470 of SEQ ID NO:l.
17. The method of claim 16 wherein said inhibitor comprises or encodes one or more of the peptides selected from the group consisting of residues 356-470, 356-397, 363-390, 397-470, 390-470, 356-376, 363-404, 363-412, 367-390, 390-412, 413-420, 413-430, 414-443, 421-470, 363-382, 366-385, 369-388, 372-391, 375-394, 378-397, 381-400, 390-409, 393-412, 417-436, 420-439, 429-448, 432-451, 435-454 and 441-460 of SEQ ID No. 1, or functionally equivalent variants, derivatives or fragments of said peptide, or a nucleic acid molecule encoding said peptide, variant, derivative or fragment.
18. The method of any one of claims 1 to 17 wherein said inhibitor is a peptide which comprises or encodes the following sequence:
SQEX1LAX2X3LAX4X5X6X7KXeAX9
wherein each of Xx to Xs is any amino acid, and wherein
X7, X8 and X9 are each any amino acid other than proline; or a functionally equivalent variant, derivative or fragment thereof .
19. The method of claim 18 wherein in said peptide
Xx is preferably arginine, glutamic acid, glutamine, threonine, phenylalanine, lysine or tyrosine, especially preferably glutamine;
X2 is preferably arginine, lysine, glycine, phenylalanine, alanine, serine or asparagine, especially preferably alanine;
X3 is preferably arginine, glutamic acid, glutamine, threonine, lysine, serine, valine, alanine or leucine, especially preferably glutamic acid;
X4 is preferably glutamic acid, glutamine, lysine, leucine, serine, aspartic acid, alanine, glycine or asparagine, especially preferably glutamic acid;
X5 is preferably tyrosine;
Xe is preferably threonine, valine, isoleucine, alanine or serine, especially preferably threonine;
X7 is preferably valine, alanine, isoleucine or leucine, preferably isoleucine or alanine, especially preferably alanine;
XB preferably isoleucine, glutamic acid or glutamine, especially preferably isoleucine; and
X9 is preferably leucine.
20. The method of claim 18 or 19 wherein said peptide comprises the following sequence: SQEX1LAX2X3LAX4YX6AKIAL .
21. The method of any one of claims 1 to 17, wherein said inhibitor comprises or encodes a peptide comprising the following sequence:
D/ ^ 2 3X4X LXg 7 EE 9 j_[] ^^Xj_2 ι3 wherein each of X_ and X2 is any amino acid, and wherein
Xi is preferably arginine, glutamine, alanine, glutamic acid or isoleucine, preferably glutamine;
X2 is preferably asparagine, isoleucine, glutamic acid or glutamine, preferably isoleucine;
X3 is glutamic acid, glutamine, valine, arginine or phenylalanine, preferably glutamine;
X4 is glutamic acid, arginine, leucine or methionine, preferably arginine,-
X5 is glycine, alanine, arginine or glutamic acid, preferably alanine;
Xs is aspartic acid, glutamine, lysine or arginine, preferably glutamine;
X7 is arginine, leucine, methionine or lysine, preferably leucine;
X8 is asparagine, glutamic acid, alanine or methionine, preferably glutamic acid;
Xg is serine, arginine, lysine or leucine, preferably arginine ,-
X10 is leucine, lysine, glutamic acid, arginine or valine, preferably lysine;
Xlx is aspartic acid, arginine or lysine, preferably arginine ,-
X12 is arginine, alanine or phenylalanine, preferably alanine; and
X13 is asparagine, glutamine, alanine, glutamic acid or tryptophan, preferably glutamine,- or a functionally equivalent variant, derivative or fragment thereof .
22. The method of claim 21 wherein said peptide comprises the sequence D/EX1X2X3X4ALQX7EEEX9X10RX12X13EE .
23. The method of any one of claims 1 to 14 wherein said inhibitor reduces or inhibits binding between ezrin and EBP50 and mimics the ezrin binding site on EBP50, preferably residues 329 to 358 of SEQ ID NO : 3 , or the EBP50 binding site on ezrin, preferably residues 1 to 286 of SEQ ID NO : 1.
24. The method of claim 23 wherein said inhibitor comprises or encodes one or more of the residues selected from the group consisting of residues 1-20, 20- 40, 40-60, 60-80, 80-100, 100-120, 120-140, 140-160, 160-180, 180-200, 200-220, 220-240, 240-260 and 260-286 of SEQ ID No. 1 and 329 to 349 and 339 to 358 of SEQ ID NO: 3 and functionally equivalent variants, derivatives or fragments of such peptides.
25. The method of any one of claims 1 to 14 wherein said inhibitor reduces or inhibits binding between EBP50 and Cbp/PAG and mimics the EBP50 binding site on Cbp/PAG, preferably residues 429 to 432 of SEQ ID NO : 4 , or the Cbp/PAG binding site on EBP50, preferably residues 11 to 97 or 149 to 236 of SEQ ID NO: 3.
26. The method of claim 25 wherein said inhibitor comprises or encodes one or more of the residues selected from the group consisting of residues 429-432 of SEQ ID No. 4 and residues 11-30, 31-50, 51-70, 71-90, 80-97, 149-170, 171-190, 191-210, 211-230 and 215-236 of SEQ ID No. 3 and functionally equivalent variants, derivatives or fragments of such peptides.
27. The method of any one of claims 1 to 26 wherein said inhibitor is a peptide, preferably consisting of less than 100 amino acids, preferably less than 50 amino acids .
28. The method of claim 27 wherein said peptide consists of 8 to 20 amino acids.
29. The method of any one of claims 1 to 28 wherein said inhibitor is a peptidomimetic derived from the sequence of a peptide as defined in any one of claims 12 or 14 to 28 .
30. The method as claimed in any one of claims 1 to 29 for in vitro or ex vivo use.
31. An inhibitor molecule which reduces or inhibits the binding between one or more of the binding partners as defined in any one of claims 1 or 6 to 9, which preferably is capable of altering the PKA type I signalling pathway in a cell .
32. The inhibitor molecule of claim 31, wherein said molecule is a peptide as defined in claim 27 or 28, or a peptidomimetic as defined as claim 29.
33. The inhibitor of claim 31 wherein said inhibitor is an antibody, or an antigen-binding fragment thereof.
34. A nucleic acid molecule encoding the peptide as defined in claim 27 or 28.
35. A vector comprising the nucleic acid molecule of claim 34.
36. A host cell containing a nucleic acid molecule or vector as defined in claim 34 or 35.
37. A method of preparing a peptide or protein inhibitor as defined in claim 32 which comprises culturing a host cell containing a nucleic acid molecule as defined in claim 34 under conditions whereby said inhibitor is expressed and recovering said inhibitor thus produced.
38. An inhibitor prepared by the method of claim 37.
39. A cell containing an inhibitor as defined in any one of claims 1 to 33.
40. A pharmaceutical composition comprising one or more inhibitors as defined in claims 31 to 33 or 38 and one or more pharmaceutically acceptable excipients and/or diluents .
41. The pharmaceutical composition of claim 40 additionally comprising one or more additional active ingredients, preferably cAMP antagonists or non- nucleoside reverse transcriptase inhibitors or one or more nucleoside reverse transcriptase inhibitors or one or more HIV protease inhibitors or one or more HAART (highly active antiretroviral therapy) .
42. The pharmaceutical composition of claim 40 or 41 for use as a medicament.
43. A method of treating or preventing a disorder or disease in which abnormal, preferably elevated, PKA type I signalling occurs in a human or non-human animal comprising administering a composition according to claim 40 or 41 or a cell as defined in claim 39 to said animal .
44. The method of claim 43 wherein said disorder is an immunosuppressive disorder, preferably HIV, AIDS or CVI, or a proliferative disease in which PKA signalling has been implicated, preferably cancer.
45. The use of the composition according to claim 40 or 41 or an inhibitor as defined in claims 31 to 33 or 38 in the manufacture of a medicament for the treatment or prevention of immunosuppressive disorders or proliferative diseases as defined in claim 44.
46. The method of any one of claims 1 to 30, 43 or 44, wherein said method is used to reverse cAMP hyperactivation that produces T cell dysfunction in immunodeficiencies by interrupting cAMP and PKA-mediated signal transduction in T cells, particularly T cell lipid rafts.
47. A method of testing the ability of a molecule to reduce or inhibit binding between any one of the binding partners as defined in claims 1 or 6 to 9 , wherein said test molecule is contacted with said binding partners and the extent of binding is assessed.
48. A product containing one or more inhibitors as defined in any one of claims 31 to 33 or 38 and one or more additional active ingredients, preferably as defined in claim 41 as a combined preparation for simultaneous, separate or sequential use in human or animal therapy.
PCT/GB2004/005396 2003-12-23 2004-12-23 Method of altering the pka type i signalling pathway WO2005060996A2 (en)

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