COMPOSITIONS FOR MOD ULA TING MRNA TRANSLA TION

FIELD OF INVENTION

[0001] The invention relates to modulation of mRNA translation. More particularly, the invention relates to modulation of initiation of mRNA translation.

BACKGROUND OF THE INVENTION

[0002] The stages involved in the translation of mRNA into protein are grouped into three phases: initiation, elongation, and termination.

[0003] The rate at which a cell synthesizes proteins is controlled primarily by the rate at which this process is initiated. Thus, any factor that alters the rate of initiation will generally produce an equivalent effect on translation of mRNA into newly synthesized protein. In eukaryotes, initiation is controlled by a family of proteins referred to as eukaryotic initiation factors (elF). Initiation involves at least 28 different polypeptides, with new proteins still being identified. At the heart of this process lie the eIF2 and eIF4 initiation factors. [0004] The eIF2 subgroup includes the heterotrimer eIF2 (subunits eIF2α, eIF2β and eIF2γ) and the heteropentamer eIF2B (subunits eIF2Bα, eIF2Bβ, eIF2Bγ, eIF2Bδ and eIF2Bε), and these proteins are required for bringing initiator methionyl-tRNA to the 4OS ribosomal subunit. More specifically, in its GTP -bound conformation, eIF2-GTP acts to promote the formation of a ternary complex involving eIF2-GTP-Met-tRNAj which then binds to the 4OS ribosomal subunit to initiate protein synthesis. The guanine nucleotide- bound state of eIF2 (i.e GDP vs. GTP) is controlled by the heteropentameric (αβδεγ) guanine nucleotide exchange factor (GEF), eIF2B.

[0005] The eIF4 subgroup includes the monomelic proteins eIF4A, eIF4B, eIF4E and eIF4G, which bring mRNA and the ribosome together and whose coordinated function confers an RNA helicase activity that is specifically targeted to the mRNA 5 '-cap structure.

[0006] Members of the eIFl and eIF5 groups primarily serve regulatory functions, while eIF3 is a 12-peptide hetero-oligomer that acts as a scaffold which coordinates interactions between the 4OS subunit and the other initiation factors. [0007] The activation of eIF2 by eIF2B is subject to regulation by a number of endogenous intracellular signaling mechanisms. For example, one of the mechanisms to regulate eIF2 activity is phosphorylation of its α subunit in response to various cellular stressors. This has been identified to be mediated by a number of stress-related kinases which all act on a conserved residue, serine 51. Additionally, phosphorylation of serine 535 on the epsilon subunit of eIF2B (eIF2Bε) by glycogen synthase kinase 3 (GSK3) has also been reported as a mechanism of controlling insulin-dependent protein synthesis.

The control of protein synthesis in response to stress, however, is multifaceted and cannot be solely explained by these phosphorylation events.

[0008] Other examples of mechanism for modulating mRNA translation have been proposed. For example, based on characterization of an interaction between eIF5 and eIF2 it has been proposed that the eIF2/eIF5 complex represents a cytoplasmic reservoir for eIF2 that antagonizes eIF2B-promoted guanine nucleotide exchange, enabling coordinated regulation of translation initiation. As a further example, a proposed mechanism for regulating cap-dependent translation has been based on disruption of eIF4E binding to the cap structure due to presence of a 4E-binding protein that can bind to eIF4E.

[0009] Protein synthesis plays a key role in cell survival, and dysregulation of the translational machinery can contribute to disease states such as cancer. Modulation of protein synthesis is a potential target to treat such diseased states. For example, protein synthesis inhibitors including ribonucleases (for triggering apoptosis), L-asparaginase (for depleting cancer cell asparagine stores), and mTOR inhibitors (i.e., mammalian target of Rapamycin inhibitors which are believed to modulate eIF4 activity) have each been proposed as anti-cancer treatments.

[0010] Abnormalities in eIF2-eIF2B interactions and/or the processes that regulate the latter can be detrimental. Alterations in the initiation of mRNA translation have been

identified in a number of disease states, and many of these are due to abnormalities in the eIF2B-dependent activation of eIF2. Pathogenic processes which appear to involve such changes include some types of tumori genesis, type 2 diabetes, some types of viral infection, and some myelination disorders. Identifying novel protein modulators of initiation may provide for new therapeutic strategies to alleviate or at least hinder the development of these human health problems.

[0011] An object of an aspect of the present invention is to provide a novel target for modulation of mRNA translation.

SUMMARY OF THE INVENTION [0012] In an aspect there is provided an isolated polypeptide that inhibits the eIF2-eIF2B interaction or inhibits mRNA translation having at least about 95% identity to SEQ ID NO: 1 or 2.

[0013] In another aspect there is provided an isolated polypeptide that inhibits a mammalian eIF2-eIF2B interaction and modulates initiation of mammalian mRNA translation.

[0014] In yet another aspect there is provided an isolated complex comprising RGS2 and at least one of the following polypeptides: RGS2, any eIF2B subunit, or eIF2. [0015] In still another aspect there is provided a method for identifying a modulator of a RGS2:eIF2Bε polypeptide complex, comprising the steps of (i) providing a reaction mixture comprising RGS2 and at least one of the following polypeptides: RGS2, any eIF2B subunit, or eIF2; (ii) contacting the reaction mixture with a test agent, and (iii) determining the effect of the test agent on the formation or stability of said complex, wherein a change in the level of formation or stability of said complex is indicative of a compound that is a modulator of a

RGS2:eIF2Bε polypeptide complex.

[0016] In still yet another aspect there is provided a method for identifying a compound that modulates a RGS2:eIF2Bε polypeptide complex, comprising:

(i) contacting RGS2 and at least one of the following polypeptides: RGS2, any eIF2B subunit, or eIF2 with a test agent under conditions in which RGS2 and at least one of the following polypeptides: RGS2, any eIF2B subunit, or eIF2 interact in the absence of the test agent to form a complex; and

(ii) determining the effect of the test agent on the formation or stability of the complex, wherein a decrease or an increase in the formation or stability of said complex in the presence of the test compound relative to the absence of the test compound indicates that the test compound modulates the RGS2:eIF2Bε polypeptide complex.

[0017] These embodiments, other embodiments, and their features and characteristics will be apparent from the description, drawings, and claims that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIGURE 1. In vitro interaction between RGS2 and eIF2Bε. Flag-tagged eIF2Bε was expressed in Sf9 cells in either its monomelic form or as part of the eIF2B holoprotein (i.e. ± eIF2Bα/eIF2Bβ/eIF2Bδ/eIF2Bγ). The precleared cell lysate was mixed with 10 μg of the indicated RGS protein. The mixture was immunoprecipitated with anti-FLAG antibody, samples run on SDS-PAGE, and transferred. Membranes were probed with anti-histidine antibody to identify co-immunoprecipitated RGS proteins (upper panel) or anti-FLAG to view immunoprecipitated eIF2Bε (lower panel) from lysate. The blots shown are representative of at least three independent experiments. [0019] FIGURE 2. Co-immunoprecipitation of endogenous RGS2 with endogenous eIF2Bε. UMR-106 osteoblast-like osteosarcoma cells were treated for 3 hours with forskolin (100 μM) and then lysed (lanes 1 -3). These lysates were incubated with protein

A/G agarose beads (lanes 1 and 2) either without (lane 1) or with (lane 2) anti-eIF2Bε, or else added directly to the gel (lane 3). Protein was removed from the beads by heating and added to the gels (lane 1 and 2). As positive IB controls, purified 10His-RGS2 (lane 4) and lysate from SfP cells infected with baculovirus encoding eIF2Bε (lane 5) were added directly to the gel. Lane 6 shows lysate acquired in parallel to that in lane 3 but using vehicle-treated cells. The SDS-PAGE gel was transferred to PVDF membrane and probed using anti-RGS2 antibody (upper panel) and then stripped and reprobed using the same eIF2Bε antibody used for the IP (lower panel). The results shown are typical of three independent experiments. [0020] FIGURE 3. Selective inhibition of in vitro protein synthesis by RGS2. (A) The effects of purified RGS proteins (4 μM) on the synthesis of the reference luciferase protein were examined in a reticulocyte-based in vitro translation. The data are presented as mean ± S.E.M of three independent experiments performed in triplicate. (B) Dose- response effects of full-length RGS proteins on steady-state, agonist-stimulated GTPase activity of Ml muscarinic receptor-activated Gαl 1. The data are presented as mean ± S.E.M of three independent experiments performed in triplicate.

[0021] FIGURE 4. RGS2-mediated inhibition of protein synthesis is independent of its RGS domain function. The effects of full-length RGS2, RGS2-N149A, and RGS2- δC169 (4 μM) were examined on (A) steady-state, agonist-stimulated GTPase activity of Ml muscarinic receptor-activated Gαl 1 or (B) synthesis of the reference luciferase protein in a reticulocyte-based in vitro translation assay. The data are presented as mean + S.E.M of three independent experiments performed in triplicate. (C) The ability of purified eIF2B to promote dissociation of [3H]GDP from purified eIF2 was examined. This activity was assessed in the absence and presence of three concentrations of RGS2, RGS2N149A, and RGS2δC169. The data are presented as mean ± S.E.M from two or more independent experiments performed in duplicate.

[0022] FIGURE 5. RGS2-mediated inhibition of protein synthesis and binding to eIF2Bε is dependent on amino acids 79- 1 16. (A) Comparison between the putative eIF2Bε- interacting domain of RGS2 and the established eIF2Bε-interacting domain of eIF2β.

Identical residues are highlighted in yellow whereas highly conserved substitutions are indicated in green. The effect of full-length RGS2, δN79-RGS2, RGS2-δC1 16, and RGS2-δ79-1 16 (4 μM) was examined on (B) steady-state, agonist-stimulated GTP ase activity of Ml muscarinic receptor-activated Gαl 1 (omitting RGS2-δC1 16, and RGS2- δ79- 1 16, which were inactive in this assay) or (C) synthesis of the reference luciferase protein in a reticulocyte-based in vitro translation assay. The data are presented as mean ± S. E. M of three independent experiments performed in triplicate. (D) RGS2 and RGS2- δ79-1 16 were examined for interactions with monomelic eIF2Bε as described for FIGURE 1. The blots shown are representative of three independent experiments. [0023] FIGURE 6. Diagram illustrating the proposed mechanism by which RGS2 inhibits the translation of mRNA into protein. The catalytic ε-subunit of the guanine nucleotide exchange factor, eIF2B, associates with the β-subunit of the GTPase, eIF2, to promote guanine nucleotide exchange on the latter. RGS2 may compete with eIF2β for binding to the same region on eIF2Bε, thereby disrupting the eIF2-eIF2B GTPase cycle, and consequently inhibiting protein synthesis.

[0024] FIGURE 7 Comparison between putative eIF2Bε-interacting domains of RGS2 and eIF2β. In this stretch of 37 amino acid residues, 24% of the amino acids are identical, and 16% are conservative substitutions (i.e., a score of >0 on the Blosum-62 matrix), indicating 40% similarity between the two proteins. The arrows denote highly nonconservative substitutions (i.e., a score of -5 on the Blosum-62 matrix). The similarity between RGS2 and eIF2β suggests that they bind competitively to eIF2Bε.

[0025] FIGURE 8. (A) Comparison of the residues between the RGS2-peptide and the RGS2 homology-peptide. (B) Both the RGS2-peptide and the RGS2 homology-peptide inhibit the translation of luciferase mRNA into new protein in a concentration-dependent manner. Full length RGS2 and RGS4 are shown as positive and negative controls, respectively.

[0026] FIGURE 9. shows decreased levels of protein synthesis in H9c2 cells infected with adenovirus coding for full-length RGS2, the RGS2 minigene, and the eIF2β

minigene compared to cells infected with a control adenovirus coding for green fluorescent protein (GFP).

DETAILED DESCRIPTION OF EMBODIMENTS

[0027] A novel role for the regulator of G protein signaling protein 2 (RGS2), a member of the RGS family of proteins is provided herein.

[0028] The regulators of G protein signaling (RGS) proteins comprise a family of at least 20 members that have been grouped into five subfamilies based on their structural and sequence homology. RGS proteins possess a conserved ~120 amino acid RGS domain that promotes their association with heterotrimeric G protein α-subunits and confers their function as GTPase-accelerating proteins (GAPs). RGS2 belongs to the B/R4 subfamily of RGS proteins that are characterized by relatively simple structures including an RGS domain that is flanked by a short amino and carboxyl terminus (with the exception of RGS3 that possesses a longer amino terminus). Although RGS2 does not appear to contain any of the established protein interacting domains that have been identified within other RGS protein sequences (e.g. PDZ, RBD, GoLoco), accumulating evidence supports the hypothesis that RGS2 may regulate cellular activity in a manner that is distinct from its known RGS domain function. For example, it has recently been shown that RGS2 interacts with the TRPV6 member of the transient receptor potential (TRP) family of cation channels to disrupt both Na ⁺ and Ca ²⁺ currents. This effect was dependent on a stretch of amino acids (1-82) situated outside of the RGS domain, as it was not observed with an amino-terminal truncation mutant, δN-RGS2. RGS2 can also bind to tubulin and enhance microtubule polymerization and this effect is dependent on a stretch of 20 amino acids located outside of its RGS domain. Moreover, research also suggests that RGS2 may have a role in regulating cAMP signaling at the level of adenylyl cyclase by interfering with the catalytic activity of this enzyme, rather than altering the rate of GTP hydrolysis by the G protein α-subunits. These studies support additional functions for RGS2 other than its role as a GAP for Ga ₁ and Gα _q proteins.

[0029] Described herein is a novel protein-protein interaction between the regulator of G protein signaling protein 2 (RGS2) and eIF2Bε (a subunit of eukaryotic initiation factor eIF2B, which conveys a rate-limiting step in protein synthesis), apparently to a site (amino acid residues 550-594 in mouse, equivalent to amino acid residues 554-598 in human) that corresponds almost exactly to its established catalytic domain (amino acid residues 552- 604 in human). The functional consequence of this novel interaction is a reduction in the ability of the cellular translation machinery to synthesize proteins de novo from mRNA molecules. This novel effect of RGS2 maps to a 37 amino acid residue stretch that found within its RGS domain (79-1 16) and, the effect is mimicked by a peptide derived from this domain (SEQ ID NO: 1

LWSEAFDELLASKYGLAAFRAFLKSEFCEENIE FWLA). Similarly, protein synthesis is inhibited by a 37 amino acid residue peptide based on the corresponding RGS2 homology domain of the β subunit of eIF2 (eIF2β) (SEQ ID NO: 2: LLAFLLAELGTSGSIDGNNQLVIKGRFQQKQIENVLR). RGS2 and the two peptides all are thought to inhibit the initiation of protein synthesis by blocking the interaction between eIF2 and eIF2B, a step required for the commencement of mRNA translation into protein.

[0030] Thus, provided is a novel drug target, eIF2Bε, and inhibitors which represent a novel class of therapeutic agents. [0031] Peptides are described herein, that may be used to modulate mRNA translation in cells without requiring introduction of genetic material into cells. Furthermore, an advantage of these peptides is that these peptides can have a direct effect on cellular function as opposed to the indirect effects of existing technologies. [0032] Accordingly, provided herein, among other things, are novel polypeptides and compositions thereof that are capable of inhibiting the initiation of protein synthesis, as well as nucleic acids, vectors, host cells, etc. for expression and production of the same. Further, provided are novel methods of treating disorders caused by alterations in the initiation of mRNA translation in a subject, using the polypeptides and compositions

thereof, optionally in conjunction with other modes of therapy, as well as kits for the practice of the same.

[0033] Also provided are methods for optimizing and identifying modulators of the eIF2 and eIF2B interaction using the novel interaction between RGS2 and eIF2Bε, as well as preparing and purifying complexes between RGS2 and eIF2Bε for use in such methods. [0034] A. Definitions

[0035] For convenience, before further description of embodiments, certain terms employed in the specification, examples and appended claims are defined here. [0036] The singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise.

[0037] "Activity" or "biological activity" or "bioactivity" or "biological function", which are used interchangeably, refer to an effector or antigenic function that is directly or indirectly performed by a polypeptide (whether in its native or denatured conformation), or by any subsequence thereof. Biological activities include, but are not limited to, binding to polypeptides, binding to other proteins or molecules, activity as a DNA binding protein, as a translation or transcription regulator, ability to bind damaged DNA, enzymatic activity, methyl transferase activity, phosphorylase or kinase activity, conformational changes, changes in intracellular localization, changes in the transcription level of the gene encoding the peptide, changes in second messenger levels, etc. An activity may be modulated by directly affecting the subject polypeptide. Alternatively, a bioactivity may be altered by modulating the level of the polypeptide, such as by modulating expression of the corresponding gene.

[0038] The term "amino acid" is intended to embrace all molecules, whether natural or synthetic, which include both an amino functionality and an acid functionality and capable of being included in a polymer of naturally-occurring amino acids. Exemplary amino acids include naturally-occurring amino acids; analogs, derivatives and congeners thereof; amino acid analogs having variant side chains; and all stereoisomers of any of any of the foregoing.

[0039] "Antibody" is intended to include whole antibodies, e.g., of any isotype (IgG, IgA, IgM, IgE, etc.), and includes fragments thereof which are also specifically reactive with a vertebrate, e.g., mammalian, protein. Antibodies may be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. Thus, the term includes segments of proteolytically-cleaved or recombinantly-prepared portions of an antibody molecule that are capable of selectively reacting with a certain protein. Non-limiting examples of such proteolytic and/or recombinant fragments include Fab, F(ab')2, Fab', Fv, and single chain antibodies (scFv) containing a V[L] and/or V[H] domain joined by a peptide linker. The scFv's may be covalently or non-covalently linked to form antibodies having two or more binding sites. Polyclonal, monoclonal or other purified preparations of antibodies and recombinant antibodies may be used to interact with the polypeptides described herein. [0040] A "combinatorial library" or "library" is a plurality of compounds, which may be termed "members," synthesized or otherwise prepared from one or more starting materials by employing either the same or different reactants or reaction conditions at each reaction in the library. In general, the members of any library show at least some structural diversity, which often results in chemical diversity. A library may have anywhere from two different members to about 10 ⁸ members or more. In certain embodiments, libraries have more than about 12, 50 and 90 members. In certain embodiments, the starting materials and certain of the reactants are the same, and chemical diversity in such libraries is achieved by varying at least one of the reactants or reaction conditions during the preparation of the library. Combinatorial libraries may be prepared in solution or on the solid phase. [0041] "Derived from" as that phrase is used herein indicates a peptide or nucleotide sequence selected from within a given sequence. A peptide or nucleotide sequence derived from a named sequence may contain a small number of modifications relative to the parent sequence, in most cases representing deletion, replacement or insertion of less than about 15%, or less than about 10%, and in many cases less than about 5%, of amino

acid residues or base pairs present in the parent sequence. In the case of DNAs, one DNA molecule is also considered to be derived from another if the two are capable of selectively hybridizing to one another.

[0042] "Derivative" refers to the chemical modification of a polypeptide sequence, or a polynucleotide sequence. Chemical modifications of a polynucleotide sequence may include, for example, replacement of hydrogen by an alkyl, acyl, or amino group. A derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule. A derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.

[0043] A "disorder caused by alterations in the initiation of mRNA translation" refers to any disease or condition caused by a deviation from the normal degree or level of initiation of mRNA translation. Disorders caused by alterations in the initiation of mRNA translation include, but are not limited to, the following: dysregulated cell growth and/or proliferation such as neoplasias, cardiac hypertrophy and failure, coronary artery restenosis, psoriasis, viral infections, diabetes, neurodegenerative disorders such as Alzheimer's disease and Parkinson's diseasee, diseases characterized by ER dysfunction, such as that caused by protein overproduction leading to ER stress, such as diabetes, Parkinson's disease, hyperhomocysteinemia, and ischemia-reperfiision and diseases specifically linked to dysfunction in eIF2B.

[0044] A "fusion protein" or "fusion polypeptide" refers to a chimeric protein as that term is known in the art and may be constructed using methods known in the art. In many examples of fusion proteins, there are two different polypeptide sequences, and in certain cases, there may be more. The sequences may be linked in frame. A fusion protein may include a domain which is found (albeit in a different protein) in an organism which also expresses the first protein, or it may be an "interspecies", "intergenic", etc. fusion expressed by different kinds of organisms. In various embodiments, the fusion polypeptide may comprise one or more amino acid sequences linked to a first polypeptide. In the case where more than one amino acid sequence is fused to a first

polypeptide, the fusion sequences may be multiple copies of the same sequence, or alternatively, may be different amino acid sequences. The fusion polypeptides may be fused to the N-terminus, the C-terminus, or the N- and C-terminus of the first polypeptide. Exemplary fusion proteins include polypeptides comprising a glutathione S- transferase tag (GST-tag), histidine tag (His-tag), an immunoglobulin domain or an immunoglobulin binding domain.

[0045] "Gene" or "recombinant gene" refers to a nucleic acid molecule comprising an open reading frame and including at least one exon and (optionally) an intron sequence. "Intron" refers to a DNA sequence present in a given gene which is spliced out during mRNA maturation.

[0046] "Gene construct" refers to a vector, plasmid, viral genome or the like which includes a "coding sequence" for a polypeptide or which is otherwise transcribable to a biologically active RNA (e.g., antisense, decoy, ribozyme, etc), may transfect cells, in certain embodiments mammalian cells, and may cause expression of the coding sequence in cells transfected with the construct. The gene construct may include one or more regulatory elements operably linked to the coding sequence, as well as intronic sequences, polyadenylation sites, origins of replication, marker genes, etc. [0047] "Host cell" refers to a cell transduced with a specified transfer vector. The cell is optionally selected from in vitro cells such as those derived from cell culture, ex vivo cells, such as those derived from an organism, and in vivo cells, such as those in an organism. "Recombinant host cells" refers to cells which have been transformed or transfected with vectors constructed using recombinant DNA techniques. "Host cells" or "recombinant host cells" are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0048] "Interact" is meant to include detectable interactions between molecules, such as may be detected using, for example, a hybridization assay. Interact also includes "binding" interactions between molecules. Interactions may be, for example, protein- protein, protein-nucleic acid, protein-small molecule or small molecule-nucleic acid in nature. Such interactions may be either direct or indirect (eg., the simultaneous binding of two proteins to a common binding partner, such as a third protein or a protein complex), and may result in changes in the function or biochemical activity of any or all interacting partners. [0049] "Isolated", with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs, or RNAs, respectively, that are present in the natural source of the macromolecule. Isolated also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Moreover, an "isolated nucleic acid" is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. "Isolated" also refers to polypeptides which are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides. [0050] "Label" and "detectable label" refer to a molecule capable of detection, including, but not limited to, radioactive isotopes, fluorophores, chemiluminescent moieties, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, ligands (e.g., biotin or haptens) and the like. "Fluorophore" refers to a substance or a portion thereof which is capable of exhibiting fluorescence in the detectable range. As an example, labels which may be used with the polypeptides, nucleic acids, peptidomimetics, or antibodies described herein include fluorescein, rhodamine, dansyl, umbelliferone, Texas red, luminol, NADPH, alpha- or beta-galactosidase and horseradish peroxidase.

[0051] The term "mammal" is known in the art, and exemplary mammals include humans, primates, bovines, porcines, canines, felines, and rodents (e.g., mice and rats).

[0052] The term "modulation", when used in reference to a functional property or biological activity or process (e.g., enzyme activity or receptor binding), refers to the capacity to either up regulate (e.g., activate or stimulate), down regulate (e.g., inhibit or suppress) or otherwise change a quality of such property, activity or process. [0053] The term "modulator" refers to a polypeptide, nucleic acid, macromolecule, complex, molecule, small molecule, compound, species or the like (naturally-occurring or non-naturally-occurring), or an extract made from biological materials such as bacteria, plants, fungi, or animal cells or tissues, that may be capable of causing modulation. Modulators may be evaluated for potential activity as inhibitors or activators (directly or indirectly) of a functional property, biological activity or process, or combination of them, (e.g., agonist, partial antagonist, partial agonist, inverse agonist, antagonist, antimicrobial agents, inhibitors of microbial infection or proliferation, and the like) by inclusion in assays. In such assays, many modulators may be screened at one time. The activity of a modulator may be known, unknown or partially known. [0054] "Nucleic acid" refers to polynucleotides such as deoxyribonucleic acid (DNA), and. where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides. ESTs, chromosomes, cDNAs, mRNAs, and rRNAs are representative examples of molecules that may be referred to as nucleic acids." Nucleic acid corresponding to a gene" refers to a nucleic acid that may be used for detecting the gene, e.g., a nucleic acid which is capable of hybridizing specifically to the gene. [0055] The term "operably linked", when describing the relationship between two nucleic acid regions, refers to a juxtaposition wherein the regions are in a relationship permitting them to function in their intended manner. For example, a control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences, such as when the appropriate molecules (e.g., inducers and polymerases) are bound to the control or regulatory sequence(s).

[0056] A "patient", "subject" or "host" to be treated by the subject method may mean either a human or non-human animal.

[0057] "Peptidomimetic" refers to a compound containing peptide-like structural elements that is capable of mimicking the biological action (s) of a natural parent polypeptide.

[0058] The phrase "pharmaceutically acceptable" refers to those compositions and dosages thereof within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0059] The phrase "pharmaceutically-acceptable carrier" means a pharmaceutically- acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any supplement or composition, or component thereof, from one organ, or portion of the body, to another organ, or portion of the body. The term "pharmaceutically acceptable carrier" refers to a carrier(s) that is "acceptable" in the sense of being compatible with the other ingredients of a composition and not deleterious to the recipient thereof. Some examples of materials which may serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen- free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[0060] "Protein", "polypeptide" and "peptide" are used interchangeably herein when referring to a gene product, e.g., as may be encoded by a coding sequence. By "gene product" it is meant a molecule that is produced as a result of transcription of a gene. Gene products include RNA molecules transcribed from a gene, as well as proteins translated from such transcripts.

[0061] The terms "polypeptide fragment" or "fragment" or "truncated polypeptide", when used in reference to a reference polypeptide, refers to a polypeptide in which amino acid residues are deleted as compared to the reference polypeptide itself, but where the remaining amino acid sequence is usually identical to the corresponding positions in the reference polypeptide. Such deletions may occur at the amino-terminus or carboxy- terminus of the reference polypeptide, or alternatively both. Fragments typically are at least 5, 6, 8 or 10 amino acids long, at least 14 amino acids long, at least 20, 30, 40 or 50 amino acids long, at least 75 amino acids long, or at least 100, 150, 200, 300, 500 or more amino acids long. A fragment can retain one or more of the biological activities of the reference polypeptide. Further, fragments can include a sub-fragment of a specific region, which sub-fragment retains a function of the region from which it is derived. [0062] The term "purified" refers to an object species that is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition). A "purified fraction" is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all species present. In making the determination of the purity of a species in solution or dispersion, the solvent or matrix in which the species is dissolved or dispersed is usually not included in such determination; instead, only the species (including the one of interest) dissolved or dispersed are taken into account. Generally, a purified composition will have one species that comprises more than about 80 percent of all species present in the composition, more than about 85%, 90%, 95%, 99% or more of all species present. The object species may be purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single species. A skilled artisan may purify a polypeptides described

herein using standard techniques for protein purification in light of the teachings herein. Purity of a polypeptide may be determined by a number of methods known to those of skill in the art, including for example, amino-terminal amino acid sequence analysis, gel electrophoresis and mass-spectrometry analysis. [0063] "Recombinant protein", "heterologous protein" and "exogenous protein" are used interchangeably to refer to a polypeptide which is produced by recombinant DNA techniques, wherein generally, DNA encoding the polypeptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous protein. That is, the polypeptide is expressed from a heterologous nucleic acid.

[0064] The term "regulatory sequence" is a generic term used throughout the specification to refer to polynucleotide sequences, such as initiation signals, enhancers, regulators and promoters, that are necessary or desirable to affect the expression of coding and non-coding sequences to which they are operably linked. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzvmology, Academic Press, San Diego, CA (1990), and include, for example, the early and late promoters of SV40, adenovirus or cytomegalovirus immediate early promoter, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda, the control regions for fd coat protein, the promoter for 3- phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast a-mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. The nature and use of such control sequences may differ depending upon the host organism. In prokaryotes, such regulatory sequences generally include promoter, ribosomal binding site, and transcription termination sequences. The term "regulatory sequence" is intended to include, at a minimum, components whose presence may influence expression, and may also include additional components whose presence is advantageous, for example,

leader sequences and fusion partner sequences. In certain embodiments, transcription of a polynucleotide sequence is under the control of a promoter sequence (or other regulatory sequence) which controls the expression of the polynucleotide in a cell-type in which expression is intended. It will also be understood that the polynucleotide can be under the control of regulatory sequences which are the same or different from those sequences which control expression of the naturally-occurring form of the polynucleotide.

[0065] The term "RGS2:eIF2Bε complex polypeptide" refers to an individual polypeptide that may be present in a RGS2:eIF2Bε complex, such as RGS2, any eIF2B subunit, or eIF2.

[0066] The term "sequence homology" refers to the proportion of base matches between two nucleic acid sequences or the proportion of amino acid matches between two amino acid sequences. When sequence homology is expressed as a percentage, e.g., 50%, the percentage denotes the proportion of matches over the length of sequence from a desired sequence (e.g., SEQ. ID NO: 1) that is compared to some other sequence. Gaps (in either of the two sequences) are permitted to maximize matching; gap lengths of 15 bases or less are usually used, 6 bases or less are used more frequently, with 2 bases or less used even more frequently. The term "sequence identity" means that sequences are identical (i.e., on a nucleotide-by-nucleotide basis for nucleic acids or amino acid-by-amino acid basis for polypeptides) over a window of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the comparison window, determining the number of positions at which the identical amino acids occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percentage of sequence identity. Methods to calculate sequence identity are known to those of skill in the art and described in further detail below.

[0067] "Small molecule" refers to a composition, which has a molecular weight of less than about 2000 kDa. Small molecules may be nucleic acids, peptides, polypeptides,

peptidomimetics, carbohydrates, lipids or other organic (carbon-containing) or inorganic molecules. As those skilled in the art will appreciate, based on the present description, libraries of chemical and/or biological mixtures, often fungal, bacterial, or algal extracts, may be screened with any of the assays described herein to identify compounds that modulate a bioactivity.

[0068] The term "specifically hybridizes" refers to detectable and specific nucleic acid binding. For example, polynucleotides, oligonucleotides and nucleic acids described herein can selectively hybridize to nucleic acid strands under hybridization and wash conditions that minimize appreciable amounts of detectable binding to nonspecific nucleic acids. Stringent conditions may be used to achieve selective hybridization conditions as known in the art and discussed herein. Generally, the nucleic acid sequence homology between the polynucleotides, oligonucleotides, and nucleic acids described herein and a nucleic acid sequence of interest will be at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or more. In certain instances, hybridization and washing conditions are performed under stringent conditions according to conventional hybridization procedures and as described further herein.

[0069] As applied to proteins, the term "substantial identity" means that two protein sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, typically share at least about 70 percent sequence identity, alternatively at least about 80, 85, 90, 95 percent sequence identity or more. In certain instances, residue positions that are not identical differ by conservative amino acid substitutions. Similarly, as applied to nucleic acids, substantial identity means that two nucleic acid sequences, when optimally aligned using default gap weights, typically share at least about 70 percent sequence identity, alternatively at least about 80, 85, 90, 95 percent sequence identity or more.

[0070] "Therapeutic agent" or "therapeutic" refers to an agent capable of having a desired biological effect on a host. Chemotherapeutic and genotoxic agents are examples of therapeutic agents that are generally known to be chemical in origin, as opposed to biological, or cause a therapeutic effect by a particular mechanism of action, respectively.

Examples of therapeutic agents of biological origin include growth factors, hormones, and cytokines. A variety of therapeutic agents are known in the art and may be identified by their effects. Certain therapeutic agents are capable of regulating red cell proliferation and differentiation. Examples include chemotherapeutic nucleotides, drugs, hormones, non-specific (non-antibody) proteins, oligonucleotides (e.g., antisense oligonucleotides that bind to a target nucleic acid sequence (e.g., mRNA sequence)), peptides, and peptidomimetics .

[0071] The term "therapeutically effective amount" refers to that amount of a modulator, drug or other molecule which is sufficient to effect treatment when administered to a subject in need of such treatment. The therapeutically effective amount will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. [0072] "Treatment" or "treating" refers to therapy, prevention and prophylaxis and particularly refers to the administration of medicine or the performance of medical procedures with respect to a patient, for either prophylaxis (prevention) or to cure the infirmity or malady in the instance where the patient is afflicted. [0073] The term "variant" includes, with respect to a parent polypeptide or nucleic acid, analogs, derivatives, fragments, truncations, mutants, deletions, substitutions, insertions, fusions and the like. Any variant may be used provided that the variant retains at least one biological or immunological activity of the parent polypeptide or nucleic acid. A parent polypeptide or nucleic acid may be mutated or changed or derivatised in any manner desired (for example, any number or combination of deletions, insertions, or substitutions) to produce a corresponding variant. [0074] The term "vector" refers to a nucleic acid capable of transporting another nucleic acid to which it has been linked. One type of vector which may be used is an episome, i.e., a nucleic acid capable of extra-chromosomal replication. Other vectors include those capable of autonomous replication and expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are

operatively linked are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer to circular double stranded DNA molecules which, in their vector form are not bound to the chromosome. Infectious expression vectors, such as recombinant baculoviruses, are used to express proteins in cultured cells. Other infectious expression vectors, such as recombinant adenoviruses and vaccinia viruses, are used as vaccines to express foreign antigens in vacinees. However, the skilled person will readily recognize other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto. [0075] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained.

[0076] B. Polypeptide and Peptidomimetic Modulators of the eIF2 and eIF2B interaction

[0077] Provided are polypeptide and peptidomimetic modulators of the eIF2-eIF2B interaction. [0078] In one embodiment, a polypeptide that inhibits the eIF2-eIF2B interaction has a sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identity to SEQ ID NO: 1 LWSEAFDELLASKYGLAAFRAFLKSEFCEENIE FWLA. [0079] In another embodiment, a polypeptide that inhibits the eIF2-eIF2B interaction has a sequence having at least about about 70%, about 75%, about 80%, about 85%, about

90%, 95%, about 96%, about 97%, about 98%, about 99% or about 100% identity to SEQ ID NO: 2: LLAFLLAELGTSGSIDGNNQLVIKGRFQQKQIENVLR. [0080] In certain embodiments, a use of RGS2 or a fragment or variant thereof in the modulation of mRNA translation is provided.

[0081] In certain other embodiments, a use of RGS2 or a fragment or variant thereof in the modulation of mRNA translation that is independent of its RGS domain GTP hydrolysis function is provided.

[0082] In another embodiment, binding of RGS2 or a fragment or variant thereof to the eukaryotic initiation factor 2B ε-subunit (eIF2Bε) and modulation of mRNA translation is provided.

[0083] In a further embodiment, selective binding of RGS2 or a fragment or variant thereof to the eukaryotic initiation factor 2B ε-subunit (eIF2Bε) and modulation of mRNA translation is provided, as RGSl and RGS4 were not found to interact with eIF2Bε or interfere with de novo protein synthesis.

[0084] In another embodiment, there is provided a peptide comprising residues of 79-116 RGS2 that interacts with eIF2Bε and modulates mRNA translation. [0085] In a further embodiment, there is provided a RGS2 or fragment or variant thereof capable of interfering with the eIF2-eIF2B GTPase cycle, which is a requisite step for the initiation of mRNA translation.

[0086] In certain embodiments, medical use of a polypeptide, or a nucleic acid encoding this polypeptide, that inhibits a mammalian eIF2-eIF2B interaction and modulates initiation of mammalian mRNA translation is provided. The polypeptide for medical use may be for example SEQ ID NO:1 or 2. With respect to medical use shorter polypeptides may be advantageous, for example polypeptides shorter than 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, or 30 amino acids in length. Thus, while full-length RGS2 is clearly shown herein to be effective, a fragment of RGS2 may be advantageous for medical use. A fragment of RGS2 may exclude one of more biological activities of full-length RGS2. For example, a fragment of RGS2 may not possess the tubulin-binding ability of full- length RGS2. As another example, a fragment of RGS2 may not possess the GTP- hydrolysis accelerating function of full-length RGS2.

[0087] In certain embodiments, the subject polypeptides may comprise a fusion protein of any of the above-described polypeptides containing at least one domain which increases its solubility and/or facilitates its purification, identification, detection, and/or

delivery. Exemplary domains, include, for example, glutathione S-transferase (GST), protein A, protein G, calmodulin-binding peptide, thioredoxin, maltose binding protein, HA, myc, poly arginine, poly His, poly His- Asp or FLAG fusion proteins and tags. Additional exemplary domains include domains that alter protein localization in vivo, such as signal peptides, type III secretion system-targeting peptides, transcytosis domains, nuclear localization signals, etc. In various embodiments, a polypeptide may comprise one or more heterologous fusions. Polypeptides may contain multiple copies of the same fusion domain or may contain fusions to two or more different domains. The fusions may occur at the N-terminus of the polypeptide, at the C-terminus of the polypeptide, or at both the N- and C-terminus of the polypeptide. Linker sequences between a polypeptide and the fusion domain may be included in order to facilitate construction of the fusion protein or to optimize protein expression or structural constraints of the fusion protein. In another embodiment, the polypeptide may be constructed so as to contain protease cleavage sites between the fusion polypeptide and polypeptide in order to remove the tag after protein expression or thereafter. Examples of suitable endoproteases, include, for example, Factor Xa and TEV proteases. [0088] In another embodiment, the subject polypeptides may be modified so that the rate of traversing the cellular membrane is increased. For example, the polypeptide may be fused to a second peptide which promotes "transcytosis," e.g., uptake of the peptide by cells. The peptide may be a portion of the HIV transactivator (TAT) protein, such as the fragment corresponding to residues 37-62 or 48-60 of TAT, portions which have been observed to be rapidly taken up by a cell in vitro (Green and Loewenstein, (1989) Cell 55: 1179- 1 188). Alternatively, the internalizing peptide may be derived from the Drosophila antennapedia protein, or homologs thereof. The 60 amino acid long homeodomain of the homeo-protejn antennapedia has been demonstrated to translocate through biological membranes and can facilitate the translocation of heterologous polypeptides to which it is coupled. Thus, polypeptides may be fused to a peptide consisting of about amino acids 42-58 of Drosophila antennapedia or shorter fragments for transcytosis (Derossi et al. (1996) J Biol Chem 271 :18188-18193: Derossi et al.

(1994) J BipiChem 269: 10444-10450; and Perez et al. (1992) J Cell Sci 102:717-722). The transcytosis polypeptide may also be a non-naturally-occurring membrane- translocating sequence (MTS), such as the peptide sequences disclosed in U.S. Patent No. 6,248,558. [0089] In another embodiment, truncated polypeptides may be prepared. Truncated polypeptides have from 1 to 20 or more amino acid residues removed from either or both the N- and C-termini. Such truncated polypeptides may prove more amenable to expression, purification or characterization than the full-length polypeptide. In addition, the use of truncated polypeptides may also identify stable and active domains of the full- length polypeptide that may be more amenable to characterization or incorporation into a pharmaceutical composition. It will be understood that the term "truncated polypeptide" is used interchangeably with "polypeptide fragment".

[0090] It is also possible to modify the structure of the polypeptides for such purposes as enhancing therapeutic or prophylactic efficacy, or stability (e.g., ex vivo shelf life, resistance to proteolytic degradation in vivo, etc.). Such modified polypeptides, when designed to retain at least one activity of the naturally-occurring form of the protein, are considered "functional equivalents" of the polypeptides described in more detail herein. Such modified polypeptides may be produced, for instance, by amino acid substitution, deletion, or addition, which substitutions may consist in whole or part by conservative amino acid substitutions.

[0091] For instance, it is reasonable to expect that an isolated conservative amino acid substitution, such as replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, will not have a major affect on the biological activity of the resulting molecule. Whether a change in the amino acid sequence of a polypeptide results in a functional homolog may be readily determined by assessing the ability of the variant polypeptide to produce a response similar to that of the wild-type protein. Polypeptides in which more than one replacement has taken place may readily be tested in the same manner.

[0092] Protein homologs may be generated combinatorially. In a representative embodiment of this method, the amino acid sequences for a population of protein homologs are aligned, optionally to promote the highest homology possible. Such a population of variants may include, for example, homologs from one or more species, or homologs from the same species but which differ due to mutation. Amino acids which appear at each position of the aligned sequences are selected to create a degenerate set of combinatorial sequences. In certain embodiments, the combinatorial library is produced by way of a degenerate library of genes encoding a library of polypeptides which each include at least a portion of potential protein sequences. For instance, a mixture of synthetic oligonucleotides may be enzymatically ligated into gene sequences such that the degenerate set of potential nucleotide sequences are expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g. for phage display). [0093] There are many ways by which the library of potential homologs may be generated from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence may be carried out in an automatic DNA synthesizer, and the synthetic genes may then be ligated into an appropriate vector for expression. One purpose of a degenerate set of genes is to provide, in one mixture, all of the sequences encoding the desired set of potential protein sequences. The synthesis of degenerate oligonucleotides is well known in the art (see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al., (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos.

Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp. 273-289; Itakura et al., (\9S4) Annu. Rev. Biochem. 53:323; Itakura et al., (1984) Science 198:1056; Ike et al., (1983) Nucleic Acid Res. 1 1 :477). Such techniques have been employed in the directed evolution of other proteins (see, for example, Scott et al., (1990) Science 249:386-390; Roberts et al., (1992) PNAS USA 89:2429-2433; Devlin et al., (1990) Science 249: 404-

406; Cwirla et al., (1990) PNAS USA 87: 6378-6382; as well as U.S. Patent Nos: 5,223,409, 5,198,346, and 5,096,815).

[0094] Alternatively, other forms of mutagenesis may be utilized to generate a combinatorial library. For example, protein homologs may be generated and isolated

from a library by screening using, for example, alanine scanning mutagenesis and the like (Ruf et al., (1994) Biochemistry 33:1565-1572; Wang et al., (1994) J. Biol. Chem. 269:3095-3099; Balint et al., (1993) Gene 137:109-118; Grodberg et al., (1993) Eur. J. Biochem. 218:597-601 ; Nagashima et al., (1993) J. Biol. Chem. 268:2888-2892; Lowman et al., (1991) Biochemistry 30:10832-10838; and Cunningham et al., (1989) Science

244:1081-1085), by linker scanning mutagenesis (Gustin et al., (1993) Virology 193:653- 660; Brown et al., (1992) MoI. Cell Biol. 12:2644-2652; McKnight et al., (1982) Science 232:316); by saturation mutagenesis (Meyers et al., (1986) Science 232:613); by PCR mutagenesis (Leung et al., (1989) Method Cell MoI Biol 1 : 1 1 - 19); or by random mutagenesis (Miller et al., (1992) A Short Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor, NY; and Greener et al., (1994) Strategies in MoI Biol 7:32-34). Linker scanning mutagenesis, particularly in a combinatorial setting, is an attractive method for identifying truncated forms of proteins that are bioactive. [0095] In certain embodiments, polypeptide fragments derived from the full-length polypeptides may be used. Fragments of the polypeptides may be produced using standard polypeptide synthesis methods as will be known to one of skill in the art. Alternatively, such polypeptide fragments, as well as the subject polypeptides, may be produced using recombinant techniques. [0096] Chemical synthesis of polypeptides may be carried out using a variety of art recognized methods, including stepwise solid phase synthesis, semi-synthesis through the conformationally-assisted re-ligation of peptide fragments, enzymatic ligation of cloned or synthetic peptide segments, and chemical ligation. Native chemical ligation employs a chemoselective reaction of two unprotected peptide segments to produce a transient thioester-linked intermediate. The transient thioester-linked intermediate then spontaneously undergoes a rearrangement to provide the full length ligation product having a native peptide bond at the ligation site. Full length ligation products are chemically identical to proteins produced by cell free synthesis. Full length ligation products may be refolded and/or oxidized, as allowed, to form native disulfide-containing protein molecules, (see e.g., U.S. Patent Nos. 6,184,344 and 6,174,530; and T. W. Muir

et al., Curr. Opin. Biotech. (1993): vol. 4, p 420; M. Miller, et al., Science (1989): vol. 246, p 1 149; A. Wlodawer, et al., Science (1989): vol. 245, p 616; L. H. Huang, et al.. Biochemistry (1991): vol. 30, p 7402; M. Schnolzer, et al., Int. J. Pept. Prot. Res. (1992): vol. 40, p 180-193; K. Rajarathnam, et al., Science (1994): vol. 264, p 90; R. E. Offord, "Chemical Approaches to Protein Engineering", in Protein Design and the Development of New therapeutics and Vaccines, J. B. Hook, G. Poste, Eds., (Plenum Press, New York, 1990) pp. 253-282; C. J. A. Wallace, et al., J. Biol. Chem. (1992): vol. 267, p 3852; L. Abrahmsen, et al., Biochemistry (1991): vol. 30, p 4151; T. K. Chang, et al., Proc. Natl. Acad. Sci. USA (1994) 91 : 12544-12548; M. Schnlzer, et al., Science (1992): vol., 3256, p 221; and K. Akaji, et al., Chem. Pharm. Bull. (Tokyo) (1985) 33: 184).

[0097] In certain embodiments, generation of mimetics, e.g. peptide or non-peptide agents, which are able to mimic binding of the authentic protein to another cellular partner are provided. Such mutagenic techniques as described above, as well as the thioredoxin system, are also particularly useful for mapping the determinants of a protein which participates in a protein-protein interaction with another protein. To illustrate, the critical residues of a protein which are involved in molecular recognition of a substrate protein may be determined and used to generate peptidomimetics that may bind to the substrate protein. The peptidomimetic may then be used as an inhibitor of the wild-type protein by binding to the substrate and covering up the critical residues needed for interaction with the wild-type protein, thereby preventing interaction of the protein and the substrate. By employing, for example, scanning mutagenesis to map the amino acid residues of a protein which are involved in binding a substrate polypeptide, peptidomimetic compounds may be generated which mimic those residues in binding to the substrate. For instance, non-hydrolyzable peptide analogs of such residues may be generated using benzodiazepine (e.g., see Freidinger et al., in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., see Huffman et al., in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substituted gama lactam rings (Garvey et al., in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden,

Netherlands, 1988), keto-methylene pseudopeptides (Ewenson et al., (1986) J. Med. Chem. 29:295; and Ewenson et al., in Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co. Rockland, IL, 1985), β-turn dipeptide cores (Nagai et al., (1985) Tetrahedron Lett 26:647; and Sato et al., (1986) J Chem Soc Perkin Trans 1 :1231), and β-aminoalcohols (Gordon et al., (1985) Biochem Biophys Res Commun 126:419; and Dann et al., (1986) Biochem Biophys Res Commun 134:71).

[0098] In certain embodiments, there is provided isolated nucleic acid sequences that encode all or a substantial portion of the amino acid sequences set forth in SEQ ID NOs 1 and 2 or other polypeptides described herein, as well as vectors, host cells, and cultures for the expression and production thereof or for gene therapy methods. [0099] Use of variant sequences that are at least 70% identical to sequences of isolated nucleic acids described herein are contemplated. [00100] Determination of sequence identity of proteins and nucleic acids by computer based methods, as well as nucleic acid hybridization techniques using high stringency conditions for determining or identifying nucleic acid sequences that share high (eg., at least 70%) sequence identity are well known to the skilled person. [00101] Stringency of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of sequence identity between the probe and hybridizable sequence, the higher the relative temperature which can be used. High stringency conditions may be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50 ⁰ C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1%) bovine serum albumin/0.1% Ficoll/0.1%

polyvinylpyrrolidone/50 niM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5xSSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C. in 0.2xSSC (sodium chloride/sodium citrate) and 50% formamide at 55 ⁰ C, followed by a high- stringency wash consisting of 0. IxSSC containing EDTA at 55°C. Hybridization and wash times should be sufficient for achieving equilibrium. [00102] Isolated nucleic acids which differ from the nucleic acids encoding polypeptides described herein due to degeneracy in the genetic code. For example, a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC are synonyms for histidine) may result in "silent" mutations which do not affect the amino acid sequence of the protein. However, it is expected that DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the polypeptides will exist. One skilled in the art will appreciate that these variations in one or more nucleotides (from less than 1% up to about 3 or 5% or possibly more of the nucleotides) of the nucleic acids encoding a polypeptide described herein may exist among a given species due to natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymorphisms may be used, for example, in embodiments described herein.

[00103] Bias in codon choice within genes in a single species appears related to the level of expression of the protein encoded by that gene. Accordingly, nucleic acid sequences which have been optimized for improved expression in a host cell by altering the frequency of codon usage in the nucleic acid sequence to approach the frequency of preferred codon usage of the host cell are provided. Due to codon degeneracy, it is possible to optimize the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide.

[00104] Expression vehicles for production of a recombinant polypeptide include plasmids and other vectors. For instance, suitable vectors for the expression of a

polypeptide include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli. In certain embodiments, the subject nucleic acid is provided in a vector comprising a nucleotide sequence encoding a polypeptide described herein, and operably linked to at least one regulatory sequence. It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed. The vector's copy number, the ability to control that copy number and the expression of any other protein encoded by the vector, such as antibiotic markers, should be considered. Such vectors may be administered in any biologically effective carrier, e.g., any formulation or composition capable of effectively transfecting cells either ex vivo or in vivo with genetic material encoding a polypeptide. Approaches include insertion of the nucleic acid in viral vectors including recombinant retroviruses, adenoviruses, adeno-associated viruses, human immunodeficiency viruses, and herpes simplex viruses- 1 , or recombinant bacterial or eukaryotic plasmids. Viral vectors may be used to transfect cells directly; plasmid DNA may be delivered alone with the help of, for example, cationic liposomes (lipofectin) or derivatized (e.g., antibody conjugated), polylysine conjugates, gramicidin S, artificial viral envelopes or other such intracellular carriers. Nucleic acids may also be directly injected. Alternatively, calcium phosphate precipitation may be carried out to facilitate entry of a nucleic acid into a cell. The subject nucleic acids may be used to cause expression and over-expression of polypeptide of interest in cells propagated in culture, e.g. to produce proteins or polypeptides. [00105] In certain embodiments, a host cell transfected with a recombinant gene in order to express a polypeptide described herein is provided. The host cell may be any prokaryotic or eukaryotic cell. For example, a gene comprising a polypeptide of interest may be expressed in bacterial cells, such as E. coli, insect cells (baculovirus), yeast, insect, plant, or mammalian cells. In those instances when the host cell is human, it may or may not be in a live subject. Other suitable host cells are known to those skilled in the art. Additionally, the host cell may be supplemented with tRNA molecules not typically

found in the host so as to optimize expression of the polypeptide. Other methods suitable for maximizing expression of the polypeptide are known to those in the art. [00106] A cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art. A polypeptide may be secreted and isolated from a mixture of cells and medium comprising the polypeptide. Alternatively, a polypeptide may be retained cytoplasmically and the cells harvested, lysed and the protein isolated. A polypeptide may be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins, including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and affinity purification with antibodies specific for particular epitopes or with the ligand of a fusion tag.

[00107] As an example, a nucleic acid encoding a polypeptide described herein is introduced into a host cell, such as by transfection or infection, and the host cell is cultured under conditions allowing expression of the polypeptide. Methods of introducing nucleic acids into prokaryotic and eukaryotic cells are well known in the art. Suitable media for mammalian and prokaryotic host cell culture are well known in the art. In some instances, the nucleic acid encoding the subject polypeptide is under the control of an inducible promoter, which is induced once the host cells comprising the nucleic acid have divided a certain number of times. For example, where a nucleic acid is under the control of a beta-galactose operator and repressor, isopropyl beta-D- thiogalactopyranoside (IPTG) is added to the culture when the bacterial host cells have attained a density of about OD600 0.45-0.60. The culture is then grown for some more time to give the host cell the time to synthesize the polypeptide. Cultures are then typically frozen and may be stored frozen for some time, prior to isolation and purification of the polypeptide.

[00108] Thus, a nucleotide sequence encoding all or part of a polypeptide described herein may be used to produce a recombinant form of a protein via microbial or eukaryotic cellular processes. Ligating the sequence into a polynucleotide construct, such as an expression vector, and transforming, infecting, or transfecting into hosts, either

eukaryotic (yeast, avian, insect or mammalian) or prokaryotic (bacterial cells), are standard procedures. Similar procedures, or modifications thereof, may be employed to prepare recombinant polypeptides by microbial means or tissue-culture technology. [00109] Other embodiments of nucleic acid sequences encoding the polypeptides described herein, as well as vectors, host cells, and cultures thereof are further described below.

[00110] In another embodiment, the nucleic acid encoding a polypeptide described herein is operably linked to a bacterial promoter, e.g., the anaerobic E. coli, NirB promoter or the E. coli lipoprotein lip promoter, described, e.g., in Inouye et al. (1985) Nucl. Acids Res. 13:3101; Salmonella pagC promoter (Miller et al., supra), Shigella ent promoter (Schmitt and Payne, J. Bacteriol. 173:816 (1991)), the tet promoter on TnIO (Miller et al., supra), or the ctx promoter of Vibrio cholera. Any other promoter recognized by the skilled person may be used. The bacterial promoter can be a constitutive promoter or an inducible promoter. An exemplary inducible promoter is a promoter which is inducible by iron or in iron-limiting conditions. In fact, some bacteria, e.g., intracellular organisms, are believed to encounter iron-limiting conditions in the host cytoplasm. Examples of iron-regulated promoters of FepA and TonB are known in the art and are described, e.g., in the following references: Headley, V. et al. (1997) Infection & Immunity 65:818; Ochsner, U.A. et al. (1995) Journal of Bacteriology 177:7194; Hunt, M.D. et al. (1994) Journal of Bacteriology 176:3944; Svinarich, D.M. and S. Palchaudhuri. (1992) Journal of Diarrhoeal Diseases Research 10:139; Prince, R.W. et al. (1991) Molecular Microbiology 5:2823; Goldberg, M.B. et al. (1990) Journal of Bacteriology 172:6863; de Lorenzo, V. et al. (1987) Journal of Bacteriology 169:2624; and Hantke, K. (1981) Molecular & General Genetics 182:288. [00111] In another embodiment, a signal peptide sequence is added to the construct, such that the polypeptide is secreted from cells. Such signal peptides are well known in the art.

[00112] In one embodiment, the powerful phage T5 promoter, that is recognized by E. coli RNA polymerase is used together with a lac operator repression module to

provide tightly regulated, high level expression or recombinant proteins in E. coli. In this system, protein expression is blocked in the presence of high levels of lac repressor. [00113] In one embodiment, the DNA is operably linked to a first promoter and the bacterium further comprises a second DNA encoding a first polymerase which is capable of mediating transcription from the first promoter, wherein the DNA encoding the first polymerase is operably linked to a second promoter. In a further embodiment, the second promoter is a bacterial promoter, such as those delineated above. In even further embodiments, the polymerase is a bacteriophage polymerase, e.g., SP6, T3, or T7 polymerase and the first promoter is a bacteriophage promoter, e.g., an SP6, T3, or T7 promoter, respectively. Plasmids comprising bacteriophage promoters and plasmids encoding bacteriophage polymerases can be obtained commercially, e.g., from Promega Corp. (Madison, Wis.) and InVitrogen (San Diego, Calif), or can be obtained directly from the bacteriophage using standard recombinant DNA techniques (J. Sambrook. E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, 1989). Bacteriophage polymerases and promoters are further described, e.g., in the following references: Sagawa, H. et al. (1996) Gene 168:37; Cheng, X. et al. (1994) PNAS USA 91 :4034; Dubendorff, J.W. and F.W. Studier (1991) Journal of Molecular Biology 219:45; Bujarski, J.J. and P. Kaesberg (1987) Nucleic Acids Research 15:1337: and Studier, F.W. et al. (1990) Methods in Enzyrnology 185:60). Such plasmids can further be modified in accordance with known methods.

[00114] In another embodiment, the bacterium further comprises a DNA encoding a second polymerase which is capable of mediating transcription from the second promoter, wherein the DNA encoding the second polymerase is operably linked to a third promoter. In a further embodiment, the third promoter is a bacteria] promoter. However, more than two different polymerases and promoters could be introduced in a bacterium to obtain high levels of transcription. The use of one or more polymerase for mediating transcription in the bacterium can provide a significant increase in the amount of polypeptide in the bacterium relative to a bacterium in which the DNA is directly under

the control of a bacterial promoter. The selection of the system to adopt will vary depending on the specific use, e.g., on the amount of protein that one desires to produce. [00115] When using a prokaryotic host cell, the host cell may include a plasmid which expresses an internal T7 lysozyme, e.g., expressed from plasmid pLysSL. Lysis of such host cells liberates the lysozyme which then degrades the bacterial membrane. [00116] Other sequences that may be included in a vector for expression in bacterial or other prokaryotic cells include a synthetic ribosomal binding site; strong transcriptional terminators, e.g., t0 from phage lambda and t4 from the rrnB operon in E. coli, to prevent read through transcription and ensure stability of the expressed polypeptide; an origin of replication, e.g., CoIEl ; and beta-lactamase gene, conferring ampicillin resistance.

[00117] Other host cells include prokaryotic host cells. Examples of prokaryotic host cells are bacteria, e.g., E. coli. Other bacteria that can be used include Shigella spp., Salmonella spp., Listeria spp., Rickettsia spp., Yersinia spp., Escherichia spp., Klebsiella spp., Bordetella spp., Neisseria spp., Aeromonas spp., Franciesella spp.,

Corynebacterium spp., Citrobacter spp., Chlamydia spp., Hemophilus spp., Brucella spp., Mycobacterium spp., Legionella spp., Rhodococcus spp., Pseudomonas spp., Helicobacter spp., Vibrio spp., Bacillus spp., and Erysipelothrix spp. Most of these bacteria can be obtained from the American Type Culture Collection (ATCC; 10801 University Blvd., Manassas, VA 20110-2209).

[00118] A number of vectors exist for the expression of recombinant proteins in yeast. For instance, YEP24, YIP5, YEP51 , YEP52, pYES2, and YRP 17 are cloning and expression vehicles useful in the introduction of genetic constructs into S. cerevisiae (see, for example, Broach et al., (1983) in Experimental Manipulation of Gene Expression, ed. M. lnouye Academic Press, p. 83). These vectors may replicate in E. coli due the presence of the pBR322 ori, and in S. cerevisiae due to the replication determinant of the yeast 2 micron plasmid. In addition, drug resistance markers such as ampicillin may be used.

[00119] In certain embodiments, mammalian expression vectors contain both prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses such as the bovine papilloma virus (BPV-I ), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells. The various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures, see Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989) Chapters 16 and 17. In some instances, it may be desirable to express the recombinant protein by the use of a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUWl), and pFastBac- derived vectors.

[00120] In another variation, protein production may be achieved using in vitro translation systems. In vitro translation systems are, generally, a translation system which is a cell-free extract comprising at least the minimum elements necessary for translation of an RNA molecule into a protein. An in vitro translation system typically comprises at least ribosomes, tRNAs, initiator methionyl-tRNAMet, proteins or complexes involved in translation, e.g., eIF2, eIF3, the cap-binding (CB) complex, comprising the cap-binding protein (CBP) and eukaryotic initiation factor 4F (eIF4F). A vaπety of in vitro translation systems are well known in the art and include commercially available kits. Examples of in vitro translation systems include eukaryotic lysates, such

as rabbit reticulocyte lysates, rabbit oocyte lysates, human cell lysates, insect cell lysates and wheat germ extracts. Lysates are commercially available from manufacturers such as Promega Corp., Madison, Wis.; Stratagene, La Jolla, Calif.; Amersham, Arlington Heights, 111.; and GIBCO/BRL, Grand Island, N. Y. In vitro translation systems typically comprise macromolecules, such as enzymes, translation, initiation and elongation factors, chemical reagents, and ribosomes. In addition, an in vitro transcription system may be used. Such systems typically comprise at least an RNA polymerase holoenzyme, ribonucleotides and any necessary transcription initiation, elongation and termination factors. An RNA nucleotide for in vitro translation may be produced using methods known in the art. In vitro transcription and translation may be coupled in a one-pot reaction to produce proteins from one or more isolated DNAs. [00121] When expression of a carboxy terminal fragment of a polypeptide is desired, i.e. a truncation mutant, it may be necessary to add a start codon (ATG) to the oligonucleotide fragment comprising the desired sequence to be expressed. It is well known in the art that a methionine at the N-terminal position may be enzymatically cleaved by the use of the enzyme methionine aminopeptidase (MAP). MAP has been cloned from E. coli (Ben-Bassat et al., (1987) J. Bacteriol. 169:751-757) and Salmonella typhimurium and its in vitro activity has been demonstrated on recombinant proteins (Miller et al., (1987) PNAS USA 84:2718-1722). Therefore, removal of an N-terminal methionine, if desired, may be achieved either in vivo by expressing such recombinant polypeptides in a host which produces MAP (e.g., E. coli or CM89 or S. cerevisiae), or in vitro by use of purified MAP (e.g., procedure of Miller et al.). [00122] In cases where plant expression vectors are used, the expression of a polypeptide may be driven by any of a number of promoters. For example, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson et al., 1984, Nature, 310:511-514), or the coat protein promoter of TMV (Takamatsu et al., 1987, EMBO J., 6:307-31 1) may be used; alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi et al., 1994, EMBO J., 3: 1671-1680; Brogue et al., 1984, Science, 224:838-843); or heat shock promoters, eg., soybean hsp 17.5-E or hsp 17.3-B

(Gurley et al., 1986, MoI. Cell. Biol., 6:559-565) may be used. These constructs can be introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors; direct DNA transformation; microinjection, electroporation, etc. For reviews of such techniques see, for example, Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology. Academic Press, New York, Section VIII, pp. 421-463; and Grierson & Corey, 1988, Plant Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9. [00123] An alternative expression system which can be used to express a polypeptide is an insect system. In one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The PGHS-2 sequence may be cloned into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). Successful insertion of the coding sequence will result in inactivation of the polyhedrin gene and production of non- occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed, (e.g., see Smith et al., 1983, L Virol, 46:584, Smith, U.S. Pat. No. 4,215,051).

[00124] In a specific embodiment of an insect system, the DNA encoding the subject polypeptide is cloned into the pBlueBacIII recombinant transfer vector (Invitrogen, San Diego, Calif.) downstream of the polyhedrin promoter and transfected into Sf9 insect cells (derived from Spodoptera frugiperda ovarian cells, available from Invitrogen, San Diego, Calif.) to generate recombinant virus. After plaque purification of the recombinant virus high-titer viral stocks are prepared that in turn would be used to infect SfP or High FiveTM (BTI-TN-5B1-4 cells derived from Trichoplusia ni egg cell homogenates; available from Invitrogen, San Diego, Calif.) insect cells, to produce large quantities of appropriately post-translationally modified subject polypeptide. Although it is possible that these cells themselves could be directly useful for drug assays, the subject polypeptides prepared by this method can be used for in vitro assays.

[0100] In another embodiment, the subject polypeptides are prepared in transgenic animals, such that in certain embodiments, the polypeptide is secreted, e.g., in the milk of a female animal.

[0101] Viral vectors may also be used for efficient in vitro introduction of a nucleic acid into a cell. Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid. Additionally, polypeptides encoded by genetic material in the viral vector, e.g., by a nucleic acid contained in the viral vector, are expressed efficiently in cells that have taken up viral vector nucleic acid. [0102] Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery system of choice for the transfer of exogenous genes in vivo, particularly into mammals. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host. A major prerequisite for the use of retroviruses is to ensure the safety of their use. particularly with regard to the possibility of the spread of wild-type virus in the cell population. The development of specialized cell lines (termed "packaging cells") which produce only replication-defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are well characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A. D. (1990) Blood 76:271). Thus, recombinant retrovirus can be constructed in which part of the retroviral coding sequence (gag, pol, env) has been replaced by nucleic acid encoding one of the antisense E6AP constructs, rendering the retrovirus replication defective. The replication defective retrovirus is then packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.) Greene Publishing

Associates, (1989), Sections 9.10-9.14, and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art. Examples of suitable packaging virus lines for preparing both ecotropic and amphotropic retroviral systems include Crip, Cre, 2 and Am. Retroviruses have

been used to introduce a variety of genes into many different cell types, including neural cells, epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381 ; Chowdhury et al. (1991) Science 254: 1802- 1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647: Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) L

Immunol. 150:4104-4115; U.S. Patent No. 4,868,116; U.S. Patent No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573). [0103] In choosing retroviral vectors as a gene delivery system for nucleic acids encoding the subject polypeptides, it is important to note that a prerequisite for the successful infection of target cells by most retroviruses, and therefore of stable introduction of the genetic material, is that the target cells must be dividing. In general, this requirement will not be a hindrance to use of retroviral vectors. In fact, such limitation on infection can be beneficial in circumstances wherein the tissue (e.g., nontransformed cells) surrounding the target cells does not undergo extensive cell division and is therefore refractory to infection with retroviral vectors. [0104] Furthermore, it has been shown that it is possible to limit the infection spectrum of retroviruses and consequently of retro viral -based vectors, by modifying the viral packaging proteins on the surface of the viral particle (see, for example, PCT publications WO93/25234, WO94/06920, and WO94/11524). For instance, strategies for the modification of the infection spectrum of retroviral vectors include: coupling antibodies specific for cell surface antigens to the viral env protein (Roux et al. (1989) PNAS 86:9079-9083; Man et al. (1992) J. Gen Virol 73:3251-3255; and Goud et al. (1983) Virology 163:251-254); or coupling cell surface ligands to the viral env proteins (Neda

et al. (1991) J Biol Chem 266:14143-14146). Coupling can be in the form of the chemical cross-linking with a protein or other variety (e.g., lactose to convert the env protein to an asialoglycoprotein), as well as by generating chimeric proteins (e.g., single- chain antibody/env chimeric proteins). This technique, while useful to limit or otherwise direct the infection to certain tissue types, and can also be used to convert an ecotropic vector in to an amphotropic vector.

[0105] Moreover, use of retroviral gene delivery can be further enhanced by the use of tissue- or cell-specific transcriptional regulatory sequences which control expression of the genetic material of the retroviral vector. [0106] Another viral gene delivery system utilizes adenovirus-derived vectors. The genome of an adenovirus can be manipulated such that it encodes a gene product of interest, but is inactive in terms of its ability to replicate in a normal lytic viral life cycle (see, for example, Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434: and Rosenfeld et al. (1992) Cell 68:143-155). Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7, etc.) are well known to those skilled in the art. Recombinant adenoviruses can be advantageous in certain circumstances in that they are capable of infecting non-dividing cells and can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld et al. (1992) cited supra), endothelial cells (Lemarchand et al. (1992) Proc. Natl. Acad. Sci. USA 89:6482-6486), hepatocytes (Herz and Gerard (1993) Proc. Natl. Acad. Sci. USA 90:2812-2816) and muscle cells (Quantin et al. (1992) Proc. Natl. Acad. Sci. USA 89:2581-2584). Furthermore, the virus particle is relatively stable and amenable to purification and concentration, and, as above, can be modified so as to affect the spectrum of infectivity. Additionally, introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA). Moreover, the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene

delivery vectors (Berkner et al, supra; Haj-Ahmand and Graham (1986) J. Virol. 57:267). Most replication-defective adenoviral vectors currently in use are deleted for all or parts of the viral El and E3 genes but retain as much as 80% of the adenoviral genetic material (see, for example, Jones et al. (1979) Cell 16:683; Berkner et al., supra; and Graham et al. in Methods in Molecular Biology, EJ. Murray, Ed. (Humana, Clifton, NJ, 1991) vol. 7. pp. 109-127). Expression of the inserted genetic material can be under control of, for example, the El A promoter, the major late promoter (MLP) and associated leader sequences, the E3 promoter, or exogenously added promoter sequences. [0107] Yet another viral vector system useful for delivery of genetic material encoding the subject polypeptides is the adeno-associated virus (AAV). Adeno-associated vims is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle. (For a review see Muzyczka et al. Curr. Topics in Micro, and Immunol. (1992) 158:97-129). It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (see for example Flotte et al. ( 1992) Am. J.

Respir. Cell. MoI. Biol. 7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlin et al. (1989) J. Virol. 62:1963-1973). Vectors comprising as little as 300 base pairs of AAV can be packaged and can integrate. Space for exogenous DNA is limited to about 4.5 kb. An AAV vector such as that described in Tratschin et al. (1985 ! MoL Cell. Biol. 5:3251-3260 can be used to introduce DNA into cells. A variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81 :6466-6470; Tratschin et al. (1985) MoI. Cell. Biol. 4:2072-2081 ; Wondisford et al. (1988) MoI. Endocrinol. 2:32- 39; Tratschin et al. (1984) J. Virol. 51 :611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).

[0108] In particular, a AAV delivery system suitable for targeting muscle tissue has been developed by Gregorevic, et al. , Nat Med. 2004 Aug;10(8):828-34. Epub 2004 JuI 25, which is able to 'home-in' on muscle cells and does not trigger an immune system

response. The delivery system also includes use of a growth factor, VEGF, which appears to increase penetration into muscles of the gene therapy agent. [0109] Other viral vector systems may be derived from herpes virus, vaccinia virus, and several RNA viruses. [0110] In addition to viral transfer methods, such as those illustrated above, non-viral methods can also be employed to cause expression of nucleic acids encoding the subject polypeptides, e.g. in a cell in vitro or in the tissue of an animal. Most nonviral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules. In certain embodiments, non- viral gene delivery systems rely on endocytic pathways for the uptake of genetic material by the targeted cell. Exemplary gene delivery systems of this type include liposomal derived systems, polylysine conjugates, and artificial viral envelopes.

[0111] In a representative embodiment, genetic material can be entrapped in liposomes bearing positive charges on their surface (e.g., lipofectins) and, optionally, which are tagged with antibodies against cell surface antigens of the target tissue (Mizuno et al. (1992) No Shinkei Geka 20:547-551 ; PCT publication WO91/06309; Japanese patent application 1047381 ; and European patent publication EP-A-43075). For example, lipofection of papilloma-infected cells can be carried out using liposomes tagged with monoclonal antibodies against PV-associated antigen (see Viae et al. (1978) J Invest Dermatol 70:263-266; see also Mizuno et al. (1992) Neurol. Med. Chir. 32:873-876). [00125] In yet another illustrative embodiment, the gene delivery system comprises an antibody or cell surface ligand which is cross-linked with a gene binding agent such as polylysine (see, for example, PCT publications WO93/04701, WO92/22635, WO92/20316, WO92/19749, and WO92/06180). For example, genetic material encoding the subject chimeric polypeptides can be used to transfect hepatocytic cells in vivo using a soluble polynucleotide carrier comprising an asialoglycoprotein conjugated to a polycation, e.g., polylysine (see U.S. Patent 5,166,320). It will also be appreciated that effective delivery of the subject nucleic acid constructs via mediated endocytosis can be improved using agents which enhance escape of the gene from the

endosomal structures. For instance, whole adenovirus or fusogenic peptides of the influenza HA gene product can be used as part of the delivery system to induce efficient disruption of DNA-comprising endosomes (Mulligan et al. (1993) Science 260-926; Wagner et al. (1992) PNAS 89:7934; and Christiano et al. (1993) PNAS 90:2122). [00126] C. Pharmaceutical Compositions and Methods of Use

[00127] In further embodiments, pharmaceutical compositions comprising a therapeutically effective amount of the polypeptides and nucleic acids described above are provided. In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. Further, devices for administering the pharmaceutical compositions may be use, for example, devices for intravenous, intraperitoneal, or subcutaneous injection

[00128] The pharmaceutical compositions may be administered by various means, depending on their intended use, as is well known in the art. For example, if compositions are to be administered orally, they may be formulated as tablets, capsules, granules, powders or syrups. Alternatively, formulations may be administered parenterally as injections (intravenous, intrathecal, intraperitoneal or subcutaneous), drop infusion preparations or suppositories. For application by the ophthalmic mucous membrane route, compounds may be formulated as eyedrops or eye ointments. These formulations may be prepared by conventional means, and, if desired, the compounds may be mixed with any conventional additive, such as an excipient, a binder, a disintegrating agent, a lubricant, a corrigent, a solubilizing agent, a suspension aid, an emulsifying agent or a coating agent.

[00129] In subject compositions, wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants may be present in the formulated agents.

[00130] Subject compositions may be suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by

any methods well known in the art of pharmacy. The amount of agent that may be combined with a carrier material to produce a single dose vary depending upon the subject being treated, and the particular mode of administration.

[00131] Methods of preparing these formulations include the step of bringing into association agents described herein with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association agents with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product. [00132] Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia), each containing a predetermined amount of a compound thereof as an active ingredient. Compounds or compositions described herein may also be administered as a bolus, electuary, or paste.

[00133] In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the coordination complex thereof is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of

capsules, tablets and pills, the compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like. [00134] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the supplement or components thereof moistened with an inert liquid diluent. Tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. [00135] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the compound, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifϊers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, com, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

[00136] Suspensions, in addition to compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[00137] Formulations for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing a coordination complex with one or more suitable non-irritating excipients or carriers comprising, for example, cocoa butter,

polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the body cavity and release the active agent. Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

[00138] Dosage forms for transdermal administration of a supplement or component includes powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active component may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required. For transdermal administration of transition metal complexes, the complexes may include lipophilic and hydrophilic groups to achieve the desired water solubility and transport properties.

[00139] The ointments, pastes, creams and gels may contain, in addition to a supplement or components thereof, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof. [00140] Powders and sprays may contain, in addition to a supplement or components thereof, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays may additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane. [00141] Compounds may alternatively be administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the compound. A non-aqueous (e.g., fluorocarbon propellant) suspension could be used. Sonic nebulizers may be used because they minimize exposing the agent to shear, which may result in degradation of the compound. [00142] Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or suspension of the compound together with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary with the

requirements of the particular compound, but typically include non-ionic surfactants (T weens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols generally are prepared from isotonic solutions. [00143] Pharmaceutical compositions suitable for parenteral administration can comprise one or more components of a supplement in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. [00144] Examples of suitable aqueous and non-aqueous carriers which may be employed in the pharmaceutical compositions described herein include water, ethanol. polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity may be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. [00145] In further embodiments, there is provided methods of modulating initiation of mRNA translation, or a disorder caused by alterations in the initiation of mRNA translation, in a subject comprising administering a pharmaceutical composition comprising a polypeptide or polynucleotide described herein to a subject. [00146] Disorders caused by alterations in the initiation of mRNA translation included, but are not limited to, the following. (1) Diseases that are characterized by, but not limited to, dysregulated cell growth and/or proliferation such as neoplasias, cardiac hypertrophy and failure, coronary artery restenosis, asthma and psoriasis. By reducing the rate at which cellular proteins are synthesized, the compositions should decrease the rates at which aberrant cells grow and/or divide in these diseases, thereby limiting disease progression. Respective cell types targeted thus would be various neoplastic cells,

cardiomyocytes in cardiac hypertrophy and failure, vascular smooth muscle cells in restenosis and asthma, and keratinocytes in psoriasis. (2) Diseases where abnormal protein expression causes cellular malfunction and/or cell death, as occurs for example in viral infections, and in pancreatic beta cells in diabetes as well as in nerve cells in neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. By blocking the production of viral proteins, the course of infections could be limited. Similarly, by blocking excess protein production or the production of abnormally processed proteins in diabetes and neurodegenerative disorders, cell malfunction and/or death would be decreased, thus limiting disease progression. (3) Diseases characterized by ER dysfunction, such as that caused by protein overproduction leading to ER stress, such as diabetes, Parkinson's disease, hyperhomocysteinemia, and ischemia-reperfusion. (4) Diseases specifically linked to dysfunction in eIF2B. One disease specifically linked to missense mutations in eIF2B is a neurodegenerative myelination defect called "vanishing white matter", where patients experience deterioration due to decreased eIF2B function.

[00147] An isolated polypeptide or nucleic acid that is capable of modulating initiation of mRNA translation may be used to treat any neoplastic cells either benign or malignant. Leiomyoma (fibroids of the uterus) and melanocy e nevi (moles) are examples of benign neoplasms. Cancer is an example of malignant neoplasia or tumor. Any type of cancer may be treated. The cancer treated may be, for example, a carcinoma such as adenocarcinomas and squamous cell carcinomas, a melanoma, a sarcoma, a leukemia, a lymphoma, or a glioma. For example, the cancer treated may be lung cancer, cervical cancer, ovarian cancer, cancer of CNS, skin cancer, prostate cancer, sarcoma, breast cancer, leukemia, colorectal cancer, head cancer, neck cancer or kidney cancer. As another example, the cancer may be small cell lung cancer, breast cancer, acute leukemia, chronic leukemia, colorectal cancer, or brain cancer. As a more specific example, the cancer may be a carcinoma. The carcinoma may be selected from small cell carcinomas, cervical carcinomas, glioma, astrocytoma, prostate carcinomas, ovarian carcinomas, melanoma, breast carcinomas, or colorectal carcinomas.

[00148] In certain embodiments, the dosage of the subject pharmaceutical compositions will generally be in the range of about 0.01 ng to about 1O g per kg body weight, specifically in the range of about 1 ng to about 0.1 g per kg, and more specifically in the range of about 100 ng to about 10 mg per kg. [00149] An effective dose or amount, and any possible effects on the timing of administration of the formulation, may need to be identified for any particular compound described herein. This may be accomplished by routine experiment as described herein, using one or more groups of animals (for example using at least 5 animals per group), or in human trials if appropriate. The effectiveness of any compound and method of treatment or prevention may be assessed by administering the supplement and assessing the effect of the administration by measuring one or more indices associated with the neoplasm of interest, and comparing the post-treatment values of these indices to the values of the same indices prior to treatment. [00150] The precise time of administration and amount of any particular compound that will yield the most effective treatment in a given patient will depend upon the activity, pharmacokinetics, and bioavailability of a particular compound, physiological condition of the patient (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage and type of medication), route of administration, and the like. The guidelines presented herein may be used to optimize the treatment, e.g., determining the optimum time and/or amount of administration, which will require no more than routine experimentation consisting of monitoring the subject and adjusting the dosage and/or timing.

[00151] While the subject is being treated, the health of the patient may be monitored by measuring one or more of the relevant indices at predetermined times during a 24-hour period. Treatment, including supplement, amounts, times of administration and formulation, may be optimized according to the results of such monitoring. The patient may be periodically reevaluated to determine the extent of improvement by measuring the same parameters, the first such reevaluation typically occurring at the end of four weeks from the onset of therapy, and subsequent

reevaluations occurring every four to eight weeks during therapy and then every three months thereafter. Therapy may continue for several months or even years, with a minimum of one month being a typical length of therapy for humans. Adjustments to the amount(s) of agent administered and possibly to the time of administration may be made based on these reevaluations.

[00152] Treatment may be initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage may be increased by small increments until the optimum therapeutic effect is attained. [00153] The combined use of several compounds described herein, or alternatively other chemotherapeutic agents, may reduce the required dosage for any individual component because the onset and duration of effect of the different components may be complimentary. In such combined therapy, the different active agents may be delivered together or separately, and simultaneously or at different times within the day. [00154] Toxicity and therapeutic efficacy of subject compounds may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 and the ED50. Compositions that exhibit large therapeutic indices are advantageous. Although compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets the compounds to the desired site in order to reduce side effects. [00155] The data obtained from the cell culture assays and animal studies may be used in formulating a range of dosage for use in humans. The dosage of any supplement, or alternatively of any components therein, can lie within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For agents described herein, the therapeutically effective dose may be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information may be used to more

accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[00156] The pharmaceutical compositions described herein can be used in combination with other therapies to modulate mRNA translation. [00157] D. Methods of Producing, Identifying, and Isolating RGS2:eIF2Bε

Complexes

[00158] In certain embodiments, methods of producing, identifying, and isolating a

RGS2:eIF2Bε complex are provided. RGS2:eIF2Bε complexes may be produced by a variety of methods. For example, RGS2:eIF2Bε complexes may be naturally-occurring or produced in a host cell comprising nucleic acids encoding RGS2:eIF2Bε complex polypeptides, or produced in vitro in a solution comprising RGS2:eIF2Bε complexes or RGS2:eIF2Bε complex polypeptides.

[00159] A variety of materials may be used as the source of potential

RGS2:eIF2Bε complex polypeptides. In one embodiment, a cellular extract or extracellular fluid may be used. The choice of starting material for the extract may be based upon the cell or tissue type or type of fluid that would be expected to contain RGS2:eIF2Bε complex polypeptides. Micro-organisms or other organisms are grown in a medium that is appropriate for that organism and can be grown in specific conditions to promote the expression of proteins that may interact with the target protein. [00160] Extracts are prepared by methods known to those of skill in the art. The extracts may be prepared at a low temperature (e.g., 4 ⁰ C) in order to retard denaturation or degradation of proteins in the extract. The pH of the extract may be adjusted to be appropriate for the body fluid or tissue, cellular, or organellar source that is used for the procedure (e.g. pH 7-8 for cytosolic extracts from mammals, but low pH for lysosomal extracts). The concentration of chaotropic or non-chaotropic salts in the extracting solution may be adjusted so as to extract the appropriate sets of proteins for the procedure. Glycerol may be added to the extract, as it aids in maintaining the stability of many proteins and also reduces background non-specific binding. Both the lysis buffer and column buffer may contain protease inhibitors to minimize proteolytic degradation of

proteins in the extract and to protect the polypeptide. Appropriate co-factors that could potentially interact with the interacting proteins may be added to the extracting solution. One or more nucleases or another reagent may be added to the extract, if appropriate, to prevent protein-protein interactions that are mediated by nucleic acids. Appropriate detergents or other agents may be added to the solution, if desired, to extract membrane proteins from the cells or tissue. A reducing agent (e.g. dithiothreitol or 2- mercaptoethanol or glutathione or other agent) may be added. Trace metals or a chelating agent may be added, if desired, to the extracting solution. [00161] Usually, the extract is centrifuged in a centrifuge or ultracentrifuge or filtered to provide a clarified supernatant solution. This supernatant solution may be dialyzed using dialysis tubing, or another kind of device that is standard in the art, against a solution that is similar to, but may not be identical with, the solution that was used to make the extract. The extract is clarified by centrifugation or filtration again immediately prior to its use in affinity chromatography. [00162] In some cases, the crude lysate will contain small molecules that can interfere with the affinity chromatography. This can be remedied by precipitating proteins with ammonium sulfate, centrifugation of the precipitate, and re-suspending the proteins in the affinity column buffer followed by dialysis. An additional centrifugation of the sample may be needed to remove any particulate matter prior to application to the affinity columns.

[00163] In an alternative embodiment, a RGS2:eIF2Bε complex polypeptides is expressed, optionally in a heterologous cell, and purified and then mixed with a potential RGS2:eIF2Bε complex polypeptide or mixture of polypeptides to identify RGS2:eIF2Bε complex formation. The potential RGS2:eIF2Bε complex polypeptide may be a single purified or semi-purified protein, or a mixture of proteins, including a mixture of purified or semi-purified proteins, a cell lysate, a clarified cell lysate, a semi-purified cell lysate, etc.

[00164] Typically, it will be desirable to immobilize a RGS2:eIF2Bε complex polypeptide or RGS2:eIF2Bε complex to facilitate separation of RGS2:eIF2Bε

complexes from uncomplexed forms of the interacting proteins, as well as to accommodate automation of the assay. The RGS2:eIF2Bε complex or RGS2:eIF2Bε complex polypeptide, or ligand, may be immobilized onto a solid support (e.g., column matrix, microtiter plate, slide, etc.). In certain embodiments, the ligand may be purified. In certain instances, a fusion protein may be provided which adds a domain that permits the ligand to be bound to a support.

[00165] In various in vitro embodiments, the set of proteins engaged in a protein- protein interaction comprises a cell extract, a clarified cell extract, or a reconstituted protein mixture of at least semi-purified proteins. By semi-purified, it is meant that the proteins utilized in the reconstituted mixture have been previously separated from other cellular or viral proteins. For instance, in contrast to cell lysates, the proteins involved in a protein-protein interaction are present in the mixture to at least about 50% purity relative to all other proteins in the mixture, and optionally may be present in greater, even 90-95%, purity. In certain embodiments of the subject method, the reconstituted protein mixture is derived by mixing highly purified proteins such that the reconstituted mixture substantially lacks other proteins (such as of cellular or viral origin) which might interfere with or otherwise alter the ability to measure activity resulting from the given protein-protein interaction. [00166] E. Methods of Detecting RGS2:eIF2Bε Complex Formation [00167] Modulation of the formation of RGS2:eIF2Bε complexes may be quantitated using, for example, detectably labeled proteins such as radiolabeled, fluorescently labeled, or enzymatically labeled polypeptides or binding partners, by immunoassay, or by chromatographic detection. Methods of preparing and identifying RGS2:eIF2Bε complexes described above may be incorporated into the detection methods.

[00168] Typically, it will be desirable to immobilize a RGS2:eIF2Bε complex polypeptide or its binding partner to facilitate separation of RGS2:eIF2Bε complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of an RGS2:eIF2Bε complex polypeptide to a binding

partner may be accomplished in any vessel suitable for containing the reactants. Examples include microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein may be provided which adds a domain that allows the protein to be bound to a matrix. For example, glutathione-S-transferase/polypeptide (GST/polypeptide) fusion proteins may be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the binding partner, e.g. an 35s-labeled binding partner, and the test compound, and the mixture incubated under conditions conducive to complex formation, e.g. at physiological conditions for salt and pH, though slightly more stringent conditions may be desired. Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly (e.g. beads placed in scintillant), or in the supernatant after the complexes are subsequently dissociated. Alternatively, the complexes may be dissociated from the matrix, separated by SDS- PAGE, and the level of RGS2:eIF2Bε complex polypeptide or binding partner found in the bead fraction quantitated from the gel using standard electrophoretic techniques such as described in the appended examples.

[00169] Other techniques for immobilizing proteins on matrices are also available for use in the subject assay. For instance, either the RGS2:eIF2Bε complex polypeptide or its binding partner may be immobilized utilizing conjugation of biotin and streptavidin. For instance, biotinylated polypeptide molecules may be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with the polypeptide may be derivatized to the wells of the plate, and polypeptide trapped in the wells by antibody conjugation. As above, preparations of a binding partner and a test compound are incubated in the polypeptide presenting wells of the plate, and the amount of complex trapped in the well may be quantitated. Exemplary methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the

binding partner, or which are reactive with the RGS2:eIF2Bε complex polypeptide and compete with the binding partner; as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the binding partner, either intrinsic or extrinsic activity. In the instance of the latter, the enzyme may be chemically conjugated or provided as a fusion protein with the binding partner. To illustrate, the binding partner may be chemically cross-linked or genetically fused with horseradish peroxidase, and the amount of RGS2:eIF2Bε complex polypeptide trapped in the RGS2:eIF2Bε complex may be assessed with a chromogenic substrate of the enzyme, e.g. 3,3'-diamino-benzadine terahydrochloride or 4-chloro-l-napthol. Likewise, a fusion protein comprising the RGS2:eIF2Bε complex polypeptide and glutathione-S-transferase may be provided, and RGS2:eIF2Bε complex formation quantitated by detecting the GST activity using 1- chloro-2,4-dinitrobenzene (Habig et al (1974) J Biol Chem 249:7130). [00170] For processes that rely on immunodetection for quantitating one of the

RGS2:eIF2Bε complex polypeptides trapped in the RGS2:eIF2Bε complex, antibodies against the RGS2:eIF2Bε complex polypeptide, such as anti-RGS2 or anti-eIF2Bε, may be used. Alternatively, the RGS2:eIF2Bε complex polypeptide to be detected in the RGS2:eIF2Bε complex may be "epitope-tagged" in the form of a fusion protein that includes, in addition to the polypeptide sequence, a second polypeptide for which antibodies are readily available (e.g. from commercial sources). For instance, the GST fusion proteins described above may also be used for quantification of binding using antibodies against the GST moiety. Other useful epitope tags include myc-epitopes (e.g., see Ellison et al. (1991) J Biol Chem 266:21 150-21 157) which includes a 10-rεsidue sequence from c-myc, as well as the pFLAG system (International Biotechnologies, Inc.) or the pEZZ-protein A system (Pharmacia, NJ). [00171] In certain in vitro embodiments of the present assay, the protein or the set of proteins engaged in a protein-protein, protein-substrate, or protein-nucleic acid interaction comprises a reconstituted protein mixture of at least semi-purified proteins. By semi-purified, it is meant that the proteins utilized in the reconstituted mixture have been previously separated from other cellular or viral proteins. For instance, in contrast

■ to cell lysates, the proteins involved in a protein-substrate, protein-protein or nucleic acid-protein interaction are present in the mixture to at least 50% purity relative to all other proteins in the mixture, and optionally may be present at 90-95% purity. In certain embodiments of the subject method, the reconstituted protein mixture is derived by mixing highly purified proteins such that the reconstituted mixture substantially lacks other proteins (such as of cellular or viral origin) which might interfere with or otherwise alter the ability to measure activity resulting from the given protein-substrate, protein- protein interaction, or nucleic acid-protein interaction. [00172] In one embodiment, the use of reconstituted protein mixtures allows more careful control of the protein-substrate, protein-protein, or nucleic acid-protein interaction conditions. Moreover, the system may be derived to favor discovery of modulators of particular intermediate states of the protein-protein interaction. For instance, a reconstituted protein assay may be carried out both in the presence and absence of a candidate agent, thereby allowing detection of a modulator of a given protein-substrate, protein-protein, or nucleic acid-protein interaction.

[00173] Assaying biological activity resulting from a given protein- substrate, protein-protein or nucleic acid-protein interaction, in the presence and absence of a candidate modulator, may be accomplished in any vessel suitable for containing the reactants. Examples include microtitre plates, test tubes, and micro-centrifuge tubes. [00174] Typically, it will be desirable to immobilize one of the RGS2:eIF2Bε complex polypeptides to facilitate separation of RGS2:eIF2Bε complexes from uncomplexed forms of one of the proteins, as well as to accommodate automation of the assay. In an illustrative embodiment, a fusion protein may be provided which adds a domain that permits the protein to be bound to an insoluble matrix. For example, protein- protein interaction component fusion proteins may be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with a potential interacting protein, e.g. an ³⁵ S-labeled polypeptide, and the test compound and incubated under conditions conducive to complex formation . Following incubation, the beads are washed to remove any unbound

interacting protein, and the matrix bead-bound radiolabel determined directly (e.g. beads placed in scintillant), or in the supernatant after the complexes are dissociated, e.g. when microtitre plate is used. Alternatively, after washing away unbound protein, the complexes may be dissociated from the matrix, separated by SDS-PAGE gel, and the level of interacting polypeptide found in the matrix-bound fraction quantitated from the gel using standard electrophoretic techniques.

[00175] In yet another embodiment, a RGS2:eIF2Bε complex polypeptide may be used to generate a two-hybrid or interaction trap assay (see also, U.S. Patent NO: 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J Biol Chem 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; and Iwabuchi et al.

(1993) Oncogene 8: 1693-1696), for subsequently detecting agents which disrupt binding of the interaction components to one another.

[00176] In particular, the method makes use of chimeric genes which express hybrid proteins. To illustrate, a first hybrid gene comprises the coding sequence for a DNA-binding domain of a transcriptional activator may be fused in frame to the coding sequence for a "bait" protein, e.g., a RGS2:eIF2Bε complex polypeptide of sufficient length to bind to a potential interacting protein. The second hybrid protein encodes a transcriptional activation domain fused in frame to a gene encoding a "fish" protein, e.g., a potential interacting protein of sufficient length to interact with the protein-protein interaction component polypeptide portion of the bait fusion protein. If the bait and fish proteins are able to interact, e.g., form a protein-protein interaction component complex, they bring into close proximity the two domains of the transcriptional activator. This proximity causes transcription of a reporter gene which is operably linked to a transcriptional regulatory site responsive to the transcriptional activator, and expression of the reporter gene may be detected and used to score for the interaction of the bait and fish proteins. The host cell also contains a first chimeric gene which is capable of being expressed in the host cell. The gene encodes a chimeric protein, which comprises (a) a DNA-binding domain that recognizes the responsive element on the reporter gene in the host cell, and (b) a bait protein (e.g., a RGS2:eIF2Bε complex polypeptide). A second

chimeric gene is also provided which is capable of being expressed in the host cell, and encodes the "fish" fusion protein. In one embodiment, both the first and the second chimeric genes are introduced into the host cell in the form of plasmids. In another embodiment, the first chimeric gene is present in a chromosome of the host cell and the second chimeric gene is introduced into the host cell as part of a plasmid. [00177] The DNA-binding domain of the first hybrid protein and the transcriptional activation domain of the second hybrid protein may be derived from transcriptional activators having separable DNA-binding and transcriptional activation domains. For instance, these separate DNA-binding and transcriptional activation domains are known to be found in the yeast GAL4 protein, and are known to be found in the yeast GCN4 and ADRl proteins. Many other proteins involved in transcription also have separable binding and transcriptional activation domains which make them useful, and include, for example, the LexA and VP 16 proteins. It will be understood that other (substantially) transcriptionally-inert DNA-binding domains may be used in the subject constructs; such as domains of ACEl, λcl, lac repressor, jun or fos. In another embodiment, the DNA-binding domain and the transcriptional activation domain may be from different proteins. The use of a LexA DNA binding domain provides certain advantages. For example, in yeast, the LexA moiety contains no activation function and has no known affect on transcription of yeast genes. In addition, use of LexA allows control over the sensitivity of the assay to the level of interaction (see, for example, the Brent et al. PCT publication WO94/10300).

[00178] In certain embodiments, any enzymatic activity associated with the bait or fish proteins is inactivated, e.g., dominant negative or other mutants of a protein-protein interaction component can be used. [00179] Continuing with the illustrative example, a RGS2:eIF2Bε complex polypeptide of the RGS2:eIF2Bε complex , if any, between the bait and fish fusion proteins in the host cell, causes the activation domain to activate transcription of the reporter gene. The method is carried out by introducing the first chimeric gene and the second chimeric gene into the host cell, and subjecting that cell to conditions under which

the bait and fish fusion proteins and are expressed in sufficient quantity for the reporter gene to be activated. The formation of a RGS2:eIF2Bε complex containing a RGS2:eIF2Bε complex polypeptide results in a detectable signal produced by the expression of the reporter gene. [00180] In still further embodiments, the RGS2:eIF2Bε complex of interest is generated in whole cells, taking advantage of cell culture techniques to support the subject assay. For example, the RGS2:eIF2Bε complex of can be constituted in a prokaryotic or eukaryotic cell culture system. Advantages to generating the RGS2:eIF2Bε complex in an intact cell includes the ability to screen for modulators of the level or activity of the RGS2:eIF2Bε complex which are functional in an environment more closely approximating that which therapeutic use of the modulator would require, including the ability of the agent to gain entry into the cell. Furthermore, certain of the in vivo embodiments of the assay are amenable to high through-put analysis of candidate agents. [00181] The RGS2:eIF2Bε complexes and RGS2:eIF2Bε complex polypeptides can be endogenous to the cell selected to support the assay. Alternatively, some or all of the components can be derived from exogenous sources. For instance, fusion proteins can be introduced into the cell by recombinant techniques (such as through the use of an expression vector), as well as by microinjecting the fusion protein itself or mRNA encoding the fusion protein. Moreover, in the whole cell embodiments of the subject assay, the reporter gene construct can provide, upon expression, a selectable marker. Such embodiments of the subject assay are particularly amenable to high through-put analysis in that proliferation of the cell can provide a simple measure of the protein- protein interaction. [00182] The amount of transcription from the reporter gene may be measured using any method known to those of skill in the art to be suitable. For example, specific mRNA expression may be detected using Northern blots or specific protein product may be identified by a characteristic stain, western blots or an intrinsic activity. In certain embodiments, the product of the reporter gene is detected by an intrinsic activity

associated with that product. For instance, the reporter gene may encode a gene product that, by enzymatic activity, gives rise to a detection signal based on color, fluorescence, or luminescence.

[00183] F. Identification of Compounds that Modulate the RGS2:eIF2Bε Complexes and Methods of Using the Same

[00184] Modulators of RGS2:eIF2Bε complexes may be identified and developed as set forth below and otherwise using techniques and methods known to those of skill in the art. The modulators described herein may elicit a change in any of the activities selected from the group consisting of (a) a change in the level of a RGS2:eIF2Bε complex, (b) a change in the activity of a RGS2:eIF2Bε complex, (c) a change in the stability of a RGS2:eIF2Bε complex, (d) a change in the conformation of a RGS2:eIF2Bε complex, (e) a change in the activity of at least one polypeptide comprising a RGS2:eIF2Bε complex, (f) a change in the conformation of at least one polypeptide comprising a RGS2:eIF2Bε complex, (g) where the reaction mixture is a whole cell, a change in the intracellular localization of a RGS2:eIF2Bε complex or a RGS2:eIF2Bε complex polypeptide thereof, (h) where the reaction mixture is a whole cell, a change the transcription level of a gene dependent on a RGS2:eIF2Bε complex, and (i) where the reaction mixture is a whole cell, a change in second messenger levels in the cell. [00185] A number of methods for identifying a molecule which modulates a RGS2:eIF2Bε complex or RGS2:eIF2Bε complex polypeptide are known in the art. For example, in one such method, a RGS2:eIF2Bε complex or RGS2:eIF2Bε complex polypeptide is contacted with a test compound, and the activity of the RGS2:eIF2Bε complex or RGS2:eIF2Bε complex polypeptide in the presence of the test compound is determined, wherein a change in the activity of the RGS2:eIF2Bε complex or RGS2:eIF2Bε complex polypeptide is indicative that the test compound modulates the activity of RGS2:eIF2Bε complex or RGS2:eIF2Bε complex polypeptide. [00186] Compounds to be tested for their ability to act as modulators of

RGS2:eIF2Bε complexes or RGS2:eIF2Bε complex polypeptides can be produced, for

example, by bacteria, yeast or other organisms (e.g. natural products), produced chemically (e.g. polypeptides and small molecules, including peptidomimetics), or produced recombinantly. Compounds for use with the above-described methods may be selected from the group of compounds consisting of lipids, carbohydrates, polypeptides, peptidomimetics, peptide-nucleic acids (PNAs), small molecules, natural products, aptamers and polynucleotides. In certain embodiments, the compound is a polynucleotide. In certain embodiments, the compound comprises a derivative or fragment of SEQ ID NO: 1 or 2. In certain embodiments, the compound may be a member of a library of compounds. [00187] A variety of assay formats will suffice and, in light of the present disclosure, those not expressly described herein will nevertheless be comprehended by one of ordinary skill in the art. Assay formats for RGS2:eIF2Bε complex formation or activity of an RGS2:eIF2Bε complex or RGS2:eIF2Bε complex polypeptides can be generated in many different forms, and include assays based on cell-free systems, e.g. purified proteins or cell lysates, as well as cell-based assays which utilize intact cells. Simple binding assays can also be used to detect agents which, by disrupting the formation of RGS2:eIF2Bε complexes, or the binding of a RGS2:eIF2Bε complex or RGS2:eIF2Bε complex polypeptide to a substrate, can serve as a modulator. Another example of an assay for a modulator of a RGS2:eIF2Bε complex polypeptide is a competitive assay that combines a RGS2:eIF2Bε complex polypeptide and a potential modulator with RGS2:eIF2Bε complex polypeptides, recombinant molecules that comprise a RGS2:eIF2Bε complex, RGS2:eIF2Bε complex, natural substrates or ligands, or substrate or ligand mimetics, under appropriate conditions for a competitive inhibition assay. RGS2:eIF2Bε complex polypeptides can be labeled, such as by radioactivity or a colorimetric compound, such that the number of molecules of a RGS2:eIF2Bε complex polypeptide bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential modulator.

[00188] Assays may employ kinetic or thermodynamic methodology using a wide variety of techniques including, but not limited to, microcalorimetry, circular dichroism,

capillary zone electrophoresis, nuclear magnetic resonance spectroscopy, fluorescence spectroscopy, and combinations thereof. Assays may also employ any of the methods for isolating, preparing and detecting RGS2:eIF2Bε complexes as described . [00189] In many drug screening programs which test libraries of compounds and natural extracts, high throughput assays are desirable in order to maximize the number of compounds surveyed in a given period of time. Assays which are performed in cell-free systems, such as may be derived with purified or semi-purified proteins or with lysates, may be used as "primary" screens in that they can be generated to permit rapid development and relatively easy detection of an alteration in a molecular target which is mediated by a test compound. Moreover, the effects of cellular toxicity and/or bioavailability of the test compound can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect of the drug on the molecular target as may be manifest in an alteration of binding affinity with other proteins or changes in enzymatic properties of the molecular target. Accordingly, potential modifiers, e.g., modulators of RGS2:eIF2Bε complexes may be detected in a cell-free assay generated by constitution of a functional RGS2:eIF2Bε complex in a cell lysate. In an alternate format, the assay can be derived as a reconstituted protein mixture which, as described below, offers a number of benefits over lysate-based assays. [00190] The activity of a RGS2:eIF2Bε complex or a RGS2:eIF2Bε complex polypeptide may be identified and/or assayed using a variety of methods well known to the skilled artisan. For example, information about the activity of non-essential genes may be assayed by creating a null mutant strain of bacteria expressing a mutant form of, or lacking expression of, a protein of interest. The resulting phenotype of the null mutant strain may provide information about the activity of the mutated gene product. Essential genes may be studied by creating a bacterial strain with a conditional mutation in the gene of interest. The bacterial strain may be grown under permissive and non-permissive conditions and the change in phenotype under the non-permissive conditions may be used to identify and/or assay the activity of the gene product.

[00191] In an alternative embodiment, the activity of a RGS2:eIF2Bε complex or

RGS2:eIF2Bε complex polypeptide may be assayed using an appropriate substrate or binding partner or other reagent suitable to test for the suspected activity. [00192] In another embodiment, the activity of a RGS2:eIF2Bε complex or RGS2:eIF2Bε complex polypeptide may be determined by assaying for the level of expression of RNA and/or protein molecules. Transcription levels may be determined, for example, using Northern blots, hybridization to an oligonucleotide array or by assaying for the level of a resulting protein product. Translation levels may be determined, for example, using Western blotting or by identifying a detectable signal produced by a protein product (e.g., fluorescence, luminescence, enzymatic activity, etc.). Depending on the particular situation, it may be desirable to detect the level of transcription and/or translation of a single gene or of multiple genes. [00193] In certain embodiments, a commercially available mRNA translation kit

(Ambion) is used to generate Coleoptera luciferase from mRNA. The luminescent signal generated is proportional to de novo luciferase synthesis and thus is taken as a measure of protein synthesis. Thus, the inhibitory effects resulting from the addition of RGS2 (or modified forms thereof) are manifested as decreases in luminescence. [00194] Moreover, in the whole cell embodiments of the subject assay, the reporter gene construct can provide, upon expression, a selectable marker. A reporter gene includes any gene that expresses a detectable gene product, which may be RNA or protein. Typically, reporter genes are those that are readily detectable. The reporter gene may also be included in the construct in the form of a fusion gene with a gene that includes desired transcriptional regulatory sequences or exhibits other desirable properties. For instance, the product of the reporter gene can be an enzyme which confers resistance to antibiotic or other drug, or an enzyme which complements a deficiency in the host cell (i.e. thymidine kinase or dihydrofolate reductase). To illustrate, the aminoglycoside phosphotransferase encoded by the bacterial transposon gene Tn5 neo can be placed under transcriptional control of a promoter element responsive to the level of a RGS2:eIF2Bε complex or RGS2:eIF2Bε complex

polypeptide present in the cell. Such embodiments of the subject assay are particularly amenable to high through-put analysis in that proliferation of the cell can provide a simple measure of inhibition of the RGS2:eIF2Bε complex or RGS2:eIF2Bε complex polypeptide. [00195] G. Antibodies

[00196] A variety of antibodies directed to RGS2:eIF2Bε complexes,

RGS2:eIF2Bε complex polypeptides or the polypeptide compositions of Section B are also provided. [00197] Antibodies may be elicited by methods known in the art. For example, a mammal such as a mouse, a hamster or rabbit may be immunized with an immunogenic form of a RGS2:eIF2Bε complexes, RGS2:eIF2Bε complex polypeptides or the polypeptide compositions of Section B (e.g., an antigenic fragment which is capable of eliciting an antibody response). Alternativelty, immunization may occur by using a nucleic acid of the acid, which presumably in vivo expresses a RGS2:eIF2Bε complexes, RGS2:eIF2Bε complex polypeptides or the polypeptide compositions of Section B giving rise to the immunogenic response observed. Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art. The progress of immunization may be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassays may be used with the immunogen as antigen to assess the levels of antibodies.

[00198] Following immunization, antisera reactive with a RGS2:eIF2Bε complexes, RGS2:eIF2Bε complex polypeptides or the polypeptide compositions of Section B may be obtained and, if desired, polyclonal antibodies isolated from the serum. To produce monoclonal antibodies, antibody producing cells (lymphocytes) may be harvested from an immunized animal and fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells. Such techniques are well known in the art, an include, for example, the hybridoma technique (originally developed by Kohler and Milstein, (1975) Nature, 256: 495-497), as the human B cell hybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72),

and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96). Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with a RGS2:eIF2Bε complexes, RGS2:eIF2Bε complex polypeptides or the polypeptide compositions of Section B and the monoclonal antibodies isolated.

[00199] In other embodiments, the antibodies described herein, or variants thereof, are modified to make them less immunogenic when administered to a subject. For example, if the subject is human, the antibody may be "humanized"; where the complimentarity determining region(s) of the hybridoma-derived antibody has been transplanted into a human monoclonal antibody, for example as described in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest et al. (1991) Biotechnology 9, 266-273. Also, transgenic mice, or other mammals, may be used to express humanized antibodies. Such humanization may be partial or complete. [00200] In another embodiment, an antibody fragment is provided. Preparation of antibody fragments may be accomplished by any number of well-known methods. In one embodiment, phage display technology may be used to generate antibody fragment selectivity components that are specific for a desired target molecule, including, for example, Fab fragments, Fv's with an engineered intermolecular disulfide bond to stabilize the V _H - V _L pair, scFvs, or diabody fragments. As an example, production of scFv antibody fragments using phage display is described below. [00201] For phage display, an immune response to a selected immunogen is elicited in an animal (such as a mouse, rabbit, goat or other animal) and the response is boosted to expand the immunogen-specific B-cell population. Messenger RNA is isolated from those B-cells, or optionally a monoclonal or polyclonal hybridoma population. The mRNA is reverse-transcribed by known methods using either a poly-A primer or murine immunoglobulin-specifϊc primer(s), typically specific to sequences adjacent to the desired V _H and VL chains, to yield cDNA. The desired V _H and VL chains are amplified by polymerase chain reaction (PCR) typically using V _H and V _L specific

primer sets, and are ligated together, separated by a linker. V _H and V _L specific primer sets are commercially available, for instance from Stratagene, Inc. of La Jolla, California. Assembled V _H -linker-V _L product (encoding an scFv fragment) is selected for and amplified by PCR. Restriction sites are introduced into the ends of the V _H -linker-V _L product by PCR with primers including restriction sites and the scFv fragment is inserted into a suitable expression vector (typically a plasmid) for phage display. Other fragments, such as an Fab' fragment, may be cloned into phage display vectors for surface expression on phage particles. The phage may be any phage, such as lambda, but typically is a filamentous phage, such as fd and M13, typically M13. [00202] In phage display vectors, the V _H -linker-V _L sequence is cloned into a phage surface protein (for M 13, the surface proteins g3p (pill) or g8p, most typically g3p). Phage display systems also include phagemid systems, which are based on a phagemid plasmid vector containing the phage surface protein genes (for example, g3p and g8p of M 13) and the phage origin of replication. To produce phage particles, cells containing the phagemid are rescued with helper phage providing the remaining proteins needed for the generation of phage. Only the phagemid vector is packaged in the resulting phage particles because replication of the phagemid is grossly favored over replication of the helper phage DNA. Phagemid packaging systems for production of antibodies are commercially available. One example of a commercially available phagemid packaging system that also permits production of soluble ScFv fragments in bacteria cells is the

Recombinant Phage Antibody System (RPAS), commercially available from Amersham Pharmacia Biotech, Inc. of Piscataway, New Jersey and the pSKAN Phagemid Display System, commercially available from MoBiTec, LLC of Marco Island, Florida. Phage display systems, their construction and screening methods are described in detail in, among others, United States Patent Nos. 5,702,892, 5,750,373, 5,821 ,047 and 6,127, 132, each of which are incorporated herein by reference in their entirety. [00203] Typically, once phage are produced that display a desired antibody fragment, epitope-speciiϊc phage are selected by their affinity for the desired immunogen and, optionally, their lack of affinity to compounds containing certain other structural

features. A variety of methods may be used for physically separating immunogen- binding phage from non-binding phage. Typically the immunogen is fixed to a surface and the phage are contacted with the surface. Non-binding phage are washed away while binding phage remain bound. Bound phage are later eluted and are used to re-infect cells to amplify the selected species. A number of rounds of affinity selection typically are used, often increasingly higher stringency washes, to amplify immunogen-binding phage of increasing affinity. Negative selection techniques also may be used to select for lack of binding to a desired target. In that case, un-bound (washed) phage are amplified. [00204] H. Kits [00205] In further embodiments, kits for treating disorders caused by alterations in the initiation of mRNA translation in a subject in need thereof are provided. For example, a kit may also comprise one or more polypeptides or nucleic acids, or a pharmaceutical composition thereof. Kit components may be packaged for either manual or partially or wholly automated practice of the foregoing methods. In other embodiments involving kits, kits are provided including compositions described herein, and optionally instructions for their use. For example, instructions may be provided for use of kit components to modulate initiation of mRNA translation. In other embodiments, a kit may further comprise controls, reagents, buffers, and/or instructions for use. Such kits may have a variety of uses, including, for example, imaging, diagnosis, therapy, and other applications.

EXEMPLIFICATION

[00206] The present invention is further illustrated by the following examples which should not be construed as limiting in any way. The contents of all cited references including literature references, issued patents, published or non published patent applications as cited throughout this application are hereby expressly incorporated by reference. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within

the skill of the art. Such techniques are explained fully in the literature. (See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes 1 and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Patent No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J.

Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N. Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor

Laboratory); , VoIs. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986) (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1986). [00207] Example 1: Modulation of mRNA Translation by Peptides that Bind to eIF2Bε

[00208] Results

[00209] The Regulators of G protein Signaling (RGS) proteins were originally identified as a family of GTPase accelerating proteins. We have discovered a novel function for RGS2 in the control of protein synthesis. RGS2 was found to bind to the eukaryotic initiation factor 2 ε-subunit (eIF2Bε) and inhibit the translation of mRNA into new protein. This effect was selective for RGS2, as RGSl and RGS4 were not found to interact with eIF2Bε or interfere with de novo protein synthesis. The ability of RGS2 to interact with eIF2Bε and inhibit protein synthesis was dependent on a subdomain within its RGS domain spanning residues 79-1 16. Moreover, RGS2 was capable of interfering with the eIF2-eIF2B GTPase cycle, which is a requisite step for the initiation of mRNA translation. Collectively, this study has identified a novel role for RGS2 in the control of protein synthesis that is independent of its RGS domain function. [00210] Physical interaction between RGS2 and eIF2Bε

[00211] Initial studies using full-length human RGS2 as bait in a yeast two-hybrid screen pointed to an interaction with eIF2Bε (4 of 21 total positives). In contrast, when RGS4 was employed as bait and screened against the same mouse brain cDNA library, eIF2Bε was not identified as a binding partner for RGS4. Based on these results implicating eIF2Bε as a putative binding partner for RGS2, we sought to confirm this interaction at the protein level and identify the physiological relevance for the association between RGS2 and eIF2Bε. Our initial studies addressed the specificity of the interaction between RGS2 and eIF2Bε using an in vitro co-immunoprecipitation assay. We mixed purified RGS2 or RGS4 with Sf9 cell lysates overexpressing eIF2Bε and examined if there was an association between the proteins. Consistent with the results from our yeast two-hybrid screen, RGS2 was able to form a complex with eIF2Bε when the two proteins were co-expressed in Sf9 cells (FIGURE 1). RGS2 was capable of interacting with eIF2Bε whether it was expressed as a monomer or as part of the pentameric eIF2B complex (i.e. ± eIF2Bα/eIF2Bβ/eIF2Bδ/eIF2Bγ) (FIGURE 1). In contrast, RGS4 did not associate with either the monomeric form of eIF2Bε or the heteropentamer (FIGURE 1 ^' ). These data demonstrate that the ε-subunit of eIF2B selectively interacts with RGS2, and imply that the other eIF2B subunits do not prevent this interaction. [00212] Next we investigated if there was an association between endogenously expressed RGS2 and eIF2Bε; therefore, we addressed whether the interaction could occur between the native proteins in vivo. Endogenous eIF2Bε was immunoprecipitated from cell lysates with anti-eIF2Bε and the immune complex was examined for RGS2 immunoreactivity. Indeed, natively expressed RGS2 was observed to co- immunoprecipitate with endogenous eIF2Bε from whole cell lysates (FIGURE 2, lane 2). These data show that the interaction between RGS2 and eIF2Bε is genuine and suggest a possible role for RGS2 in the control of protein synthesis.

[00213] Selective inhibition of protein synthesis by RGS2

[00214] The role of eIF2B in the control of protein synthesis is to promote guanine nucleotide exchange on the eIF2 γ-subunit. In the absence of a functional eIF2-eIF2B

relationship, the normal translation of mRNA into protein cannot be initiated. Thus, we hypothesized that RGS2 may act to compete with eIF2 for binding to eIF2B and interfere with de novo protein synthesis. We monitored the production of the luminescent protein, Coleoptera luciferase, from its mRNA in the absence and presence of RGSl, RGS2, and RGS4 using an in vitro translation assay. The addition of RGS2 to the translation assay resulted m a significant decrease in protein synthesis compared to control (FIGURE 3A). In contrast, neither RGSl nor RGS4 had an effect on the synthesis of the reference luciferase protein, even at a saturating concentration for their GAP activity towards Got| ] (FIGURE 3) (see below). These data are consistent with observations that RGS2 appears to selectively associate with eIF2Bε. As a reference to confirm the integrity of the RGS proteins studied, we also examined the effects of RGSl , RGS2, and RGS4 on the GTPase activity of Gαπ. All three RGS proteins tested were able to dose-dependently increase the rate of Mi muscarinic receptor-stimulated Gαπ GTP hydrolysis to approximately the same extent (FIGURE 3B). Collectively, these results demonstrate that RGS2 interferes with the translation of mRNA into protein in a selective manner.

[00215] RGS2 regulation of protein synthesis and the RGS domain

[00216] Our next point of interest was to determine whether the RGS2-mediated inhibition of protein synthesis is dependent on having an intact RGS domain. For these studies, we generated two mutations within this region of the protein that would act to impair its GTPase accelerating function: RGS2-N 149A and RGS2-δC 169. The N 149A point mutation is thought to disrupt a critical contact point between RGS2 and G protein α-subunits, whereas δC169 removes a substantial portion of the RGS domain at its carboxyl-terminus. When the two RGS2 mutants were examined for GAP activity in our membrane-based GAP assay, neither of the RGS2 mutants was able to increase agonist- stimulated GTP hydrolysis above that of agonist alone (FIGURE 4A). Wild-type RGS2, serving as a positive control, produced a significant increase in GTP hydrolysis (FIGURE 4A). These results are consistent with the logic that in the absence of a proper RGS-Gα interaction, GTP hydrolysis cannot be accelerated. In contrast, when the two RGS2 mutants were employed in the in vitro translation assay to investigate their effects on

protein synthesis, it was observed that both proteins retained the ability to inhibit translation of luciferase mRNA into protein to the same degree as that of wild-type RGS2 (FIGURE 4B). These results show that the ability of RGS2 to inhibit protein synthesis is not dependent on its RGS domain function, per se, and suggest the mechanism is G protein-independent; therefore, we hypothesized that RGS2 may disrupt the eIF2-eIF2B GTP cycle by binding to eIF2Bε. Thus, we examined the effects of RGS2 on the GEF activity of eIF2B towards eIF2. Purified eIF2B was incubated with eIF2 that had been pre-loaded with [ ³ H]GDP and the potential for eIF2B to promote the dissociation of [ ³ H]GDP from eIF2 was measured in the absence and presence of RGS2, RGS2-N149A, and RGS2-δC169. Surprisingly, all three of the RGS2 proteins examined were able to dose-dependently inhibit eIF2B GEF activity for eIF2 (FIGURE 4C). These data show that RGS2 can interfere with eIF2B GEF activity in an RGS domain-independent manner. Moreover, the data suggest that RGS2 may function to inhibit protein synthesis by impeding eIF2B-mediated guanine nucleotide exchange on eIF2. [00217] Identification of the RGS2 domain involved in regulating protein synthesis

[00218] The interaction between RGS2 and the eIF2B heteropentamer (αβδεγ) suggests that RGS2 associates with eIF2Bε at a site(s) that is distinct from where it makes contact with the other eIF2B subunits. The observation that RGS2 can also interfere with the eIF2-eIF2B GTP cycle suggests that RGS2 may bind to eIF2Bε in a manner similar to that of eIF2β. Thus we compared the residues of eIF2β (200-333) that confer binding to eIF2B with the protein sequence of RGS2 and identified a stretch of 37 amino acids with 46 % sequence homology (FIGURE 5A); 9 of the 37 amino acids are identical (24%) and 8 out of the 37 are highly conserved substitutions (22%). We used this homologous sequence of 37 amino acids as a point of reference to generate δN79- RGS2, RGS2-δC1 16, and RGS2-δ79-116 in our attempts to identify the active domain(s) within RGS2 that is responsible for inhibiting protein synthesis. Deletion of the amino terminus of RGS2 did not interfere with RGS2 GAP activity, as was observed with the δN79-RGS2 mutant (FIGURE 5B). Agonist-stimulated Ga, , GTP hydrolysis was comparable in the presence of both wild-type RGS2 and δN79-RGS2 (FIGURE 5B).

This result is consistent with the fact that the δN79 truncation of RGS2 involves residues that are upstream of the RGS domain that do not interfere with the RGS box itself. RGS2-δC116 and RGS2-δ79-116, however, ablated RGS2 GAP activity (data not shown). This was expected given that these two mutations act to disrupt the integrity of the RGS domain. The three RGS2 deletion mutants were then used in the in vitro translation assay to determine if they had any effect on protein synthesis. Truncation of RGS2 either upstream or downstream of the homologous 37 amino acid sequence did not disrupt the ability of RGS2 to impair protein synthesis (FIGURE 5C). RGS2-δC116 and δN79-RGS2 each inhibited mRNA translation to a comparable level as that of wild-type RGS2 (FIGURE 5C). Surprisingly, when RGS2-δ79-116 was used in the translation assay, the ability of RGS2 to inhibit mRNA translation was completely blocked. Moreover, RGS2-δ79-1 16 failed to interact with monomelic eIF2Bε (FIGURE 5D). These data demonstrate that RGS2 amino acids 79-116 are both sufficient and necessary for binding to eIF2Bε and inhibiting protein synthesis. [00219] Discussion

[00220] The present results outline a novel G protein-independent function for

RGS2 in the control of protein synthesis. RGS2 is able to bind to the ε-subunit of the guanine nucleotide exchange factor, eIF2B, and this interaction appears to interfere with the initiation of mRNA translation by preventing guanine nucleotide exchange on eIF2. In contrast, the closely related RGS2 homologues, RGSl and RGS4, had no effect on protein synthesis. The results of this study suggest that RGS2 may function to maintain protein homeostasis within the cell, in addition to its established role as a GAP for heterotrimeric G proteins. [00221] A potential link between G protein-mediated signaling networks and cellular control of protein synthesis has previously been implicated, although no effects on mRNA translation have been established. The α-subunit of eIF2B has been reported to interact with the carboxyl tails of the α2A, α2B, α2C, and β2 adrenergic receptors in a yeast two-hybrid screen, but not to the carboxyl tail of the vasopressin receptor. Similarly, eIF2Bα has been shown to interact with the third intracellular loop of the α2B

and α2C adrenergic receptors in a manner that may be dependent on 14-3-3ζ. The third intracellular loop of the M4 muscarinic receptor, but not Ml or M2, was also reported to associate with eukaryotic elongation factor 1 A2 to promote guanine nucleotide exchange on the latter, which was suggested to be a mechanism of regulating M4 muscarinic receptor recycling. No effects of RGS proteins on mRNA translation have been reported until now, although RGSl was identified as a binding partner for eukaryotic initiation factor 3δ in a yeast two-hybrid screen (www.signaling-gateway.org/data/Y2H/cgi- bin/y2h_int.cgi?id=l 7628). The latter interaction was not confirmed at the protein level and our study has not determined RGSl to have any effect on protein synthesis. Nonetheless, these data support a role for G protein-mediated signaling networks in the control of protein synthesis.

[00222] Under physiological conditions protein synthesis occurs on a "demand and supply" basis; however, under conditions of cellular stress such as oxidative damage, nutrient deprivation, viruses, and heat shock, the overall rate at which mRNA is translated into protein is reduced (although the synthesis of stress-related proteins is maintained or increased through specialized alternative pathways). A variety of mechanisms exist to reduce global protein synthesis, and for the most part these involve changes in initiation. Of particular importance is the rate-limiting eIF2-eIF2B interaction, which depends upon both the activity and the availability of the exchange factor eIF2B. The best characterized inhibitory mechanism involves the phosphorylation of Ser51 on the eIF2α subunit. This is mediated by four stress-activated kinases: haem- regulated inhibitor (HRI), general control non-derepressible-2 (GCN2), protein kinase activated by double-stranded RNA (PKR), and pancreatic endoplasmic reticulum eIF2α kinase (PERK). The phosphorylation of this highly conserved serine acts to decrease the dissociation rate of eIF2 from eIF2B, which essentially converts eIF2 from a substrate into a competitive inhibitor of eIF2B GEF activity. Other mechanisms for inhibiting mRNA translation by way of eIF2B have also been reported. For example, GSK3 directly phosphorylates eIF2Bε at serine 540 in intact cells and this inhibits eIF2B GEF activity by up to 80 %. In addition, it has been shown that the effects of eIF2B on eIF2

can be decreased by the binding of the latter to eIF5. The primary function of eIF5 is to promote GTP hydrolysis by eIF2, however, when present at elevated levels eIF5 may act to sequester eIF2 from eIF2B, thereby impeding protein synthesis. The present results suggest a comparable inhibitory mechanism, wherein the interaction between eIF2 and eIF2B again is impeded by the binding of a third protein, in this case the association of RGS2 with eIF2B. Although the specific protein target differs, the functional consequence in both cases is an attenuation of guanine nucleotide exchange on eIF2. [00223] One particularly interesting aspect of RGS2 signaling is that RGS2 mRNA and protein expression can be upregulated in response to various forms of cellular stress including heat shock, oxidative stress, DNA damage, and mechanical stress. For example, treatment of 1321N1 astrocytoma cells with H ₂ O ₂ leads to the upregulation of RGS2 mRNA and protein in a concentration-dependent manner, and subjecting these cells to heat shock also results in increased levels of RGS2 mRNA in a time-dependent manner. Likewise, when SH-SY5Y cells were treated with the DNA-damaging agent camptothecin, RGS2 mRNA levels were found to increase whereas RGS4 mRNA levels decreased. These studies strongly suggest that tonic changes in RGS2 expression may help to maintain cellular integrity. Indeed, RGS2 has previously been shown to act as a negative regulator of αl -adrenergic receptor-stimulated cardiomyocyte hypertrophy. When primary cultures of ventricular myocytes were treated with the αl -adrenergic receptor agonist, phenylephrine, to induce cardiomyocyte hypertrophy, RGS2 mRNA was selectively increased over RGSl, 3, 4, and 5 in a time-dependent manner. The overexpression of RGS2 in these cells completely blocked the agonist-dependent increase in cell size, as well as the expression of various genetic markers for cardiac hypertrophy. These results implicate RGS2 as having a protective role in cardiomyocyte hypertrophy, and are consistent with previous reports that downregulation of endogenous RGS2 by way of RNAi exacerbates cardiomyocyte hypertrophy. Such findings are typically thought to reflect the negative effects of RGS2 on G _q -mediated signaling; however, given that hypertrophy is characterized by an increase in protein synthesis and cell size, one could argue based on the present results that stress-induced upregulation of RGS2

expression may impede the development of cardiac hypertrophy by inhibiting global protein synthesis.

[00224] The accruing evidence to date supporting a role for RGS2 in the pathology of various cardiovascular and CNS diseases makes it a potential therapeutic target. Attempts to modulate RGS2 activity using traditional chemical small molecule modulators may manifest as unsuccessful due to a number of complicating factors including the widespread distribution of RGS2 in many tissues and the multiple mechanisms of action now associated with RGS2. The latter include the abilities of RGS2 to function as a GAP and functional antagonist for G _αq and Gα _s , respectively, and also to bind to and regulate the activities of other proteins including adenylyl cyclase, tubulin, the cation channel TRPV6, and now also eIF2B as we have shown in this report. Although to some extent these various functions involve overlapping domains within RGS2, it may be possible to selectively mimic or inhibit them pharmacologically. Our discovery that RGS2 amino acids 79-1 16 are necessary for its interaction with eIF2Bε and inhibition of mRNA translation provides a novel therapeutic target for treating diseases characterized by impaired protein synthesis such as neoplasias, diabetes, cardiac hypertrophy, and neurodegeneration. Efforts are currently underway in our laboratory to design and develop peptidomimetics to modulate this interaction. In conclusion, we provide evidence in support of a model whereby RGS2 binds to eIF2Bε to disrupt the initiation of mRNA translation during protein synthesis (FIGURE 6). [00225] Methods

[00226] Reagents and clones

[00227] Plasmids encoding histidine-tagged wild-type RGS proteins and δN79-

RGS2 were a gift from Dr. John Hepler (Emory University, Atlanta, GA). The baculovirus coding for a M] muscarinic receptor-Get] j fusion protein was provided by Dr. Tatsuya Haga (University of Tokyo, Hongo, Japan). Baculoviruses encoding all five Flag-tagged subunits of eIF2B were generated as described previously. RGS2-N149A, RGS2-δC116, and RGS2-δC169 were made using the Quikchange site-directed mutagenesis kit (Stratagene, La Jolla ,CA). RGS2 δ79-1 16 was made by inverse PCR

using phosphorothioate-modified primers as described previously. Anti-eIF2Bε and protein A/G PLUS-agarose beads were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-RGS2 was purchased from Genway Biotech, Inc. (San Diego, CA). Protein G-agarose beads were purchased from Amersham Biosciences (Piscataway, NJ). Anti-FLAG M2 monoclonal antibody and other reagents were purchased from Sigma-Aldrich Canada (Oakville, ON). [00228] Cell Culture

[00229] The UMR- 106 osteoblastic cell line was obtained from the American Type

Culture Collection (Manassas, VA) and grown in Minimum Essential Medium (MEM) alpha medium supplemented with 10 % fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B. Cells were grown in a humidified incubator in the presence of 5 % CO ₂ at 37°C. [00230] RGS Protein Purification

[00231] Histidine-tagged RGSl and RGS4 proteins were expressed in Escherichia CoIi BL21 (DE3) strain and purified to >95 % purity by nickel chromatography followed by gel filtration chromatography using a Superdex 75 HR 10/30 column (Amersham Biosciences, Piscataway, NJ) as described previously. Histidine-tagged RGS2 and all its derivative mutants were purified from bacterial inclusion bodies as follows: two liters of BL21 (DE3) bacterial culture were incubated with vigorous shaking at 37°C until mid- log phase (optical density at 600nm of 0.7). Induction was commenced by the addition of 1 mM isopropylthio-β-D-galactoside for 3-4 hr at 37°C. The bacteria were harvested by centrifugation and pellets were stored at -80°C. Cells were then thawed and resuspended in 30 ml IB buffer (20 mM Tris-HCl, pH 8.0, 10 mM EDTA, 0.1 mM PMSF, 10 μg/ml leupeptin, 1 μg/ml aprotinin, 1 % Triton X-100) and 0.2 mg/ml lysozyme was added. The suspension was mixed and incubated at 30 ⁰ C for 15 min followed by sonication on ice. The cell lysates were then centrifuged at 15 00Og for 30 min at 4 ⁰ C and the pellet was washed by resuspension and re-centrifugation once with IB buffer and once with 0.1 M Tris-HCl, pH 8.0. The protein refolding process was accomplished by extracting inclusion bodies with extraction buffer (10 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 6M

guanidine hydrochloride) followed by dialysis against buffer A (50 mM Hepes, pH 7.5, 500 mM NaCl, 20 mM β-mercaptoethanol, 10 % glycerol, 0.5 mM PMSF, 2 M urea) and then buffer B (50 mM Hepes, pH 7.5, 500 mM NaCl, 20 mM β-mercaptoethanol, 10 % glycerol, 0.5 mM PMSF, 500 mM urea). The refolded protein was then purified by nickel chromatography followed by size-exclusion chromatography as described for histidine-tagged RGSl and RGS4 except that the concentration of NaCl was 0.5 M throughout the imidazole elution step and then reduced to 0.3 M for the final gel filiation step. Protein samples were placed in aliquots and stored at -8O ⁰ C. [00232] GTP Hydrolysis Assay [00233] Sf9 cells were infected with baculoviruses encoding Gβ], Gγ ₂ , and an M 1 muscarinic receptor-Gcti _\ fusion protein and prepared for the GTP hydrolysis assay as described previously. Membranes were assayed for carbachol-stimulated GTP hydrolysis for 5 minutes at 3O ⁰ C in the absence and presence of the indicated RGS proteins. The reaction buffer contained: [γ- ³² P]GTP (1 x 10 ⁶ cpm/assay), 20 mM Hepes, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, 0.5 mM ATP, 0.1 mM PMSF, 1 μg/ml aprotinin, 10 μg/ml leupeptin, 0.1 mM ascorbic acid , 300 mM NaCl, and 2 mM MgCl ₂ . Non-specific GTPase activity was defined as that in the presence of membranes plus the inverse agonist, tropicamide (10 μM), and these values were subtracted to yield the specific agonist- and receptor-dependent signal. GTP hydrolysis reactions were terminated by the addition of 5 % Norit in 50 mM NaH ₂ PO ₄ , pH 3.0. The reaction mixture was centrifuged at 2000g and P ₁ was recovered from the supernatant. Radioactivity was counted on a Packard Tri-Carb 2900TR liquid scintillation counter (Perkin Elmer, Boston, MA) and the data were analyzed as described previously. [00234] In vitro Translation Assay [00235] Translation was measured as the synthesis of luciferase protein from luciferase mRNA using an in vitro translation kit (Ambion Inc., Austin, TX) according to the manufacturer's protocol. Briefly, rabbit reticulocyte lysates were combined with 20 μM amino acid mixture, 0.5 μg luciferase RNA (Promega, Madison, WI), IX low salt translation mix, and RGS proteins where indicated. The mixture was incubated at 30 ⁰ C

for 45 min and luminescence was detected with luciferase assay substrate (Promega, Madison, WI) using the LMax II microplate reader (Molecular Devices Corporation, Sunnyvale, CA). [00236] eIF2B GEF Assay [00237] The guanine nucleotide exchange activity of eIF2B was measured as described previously. Briefly, eIF2 and eIF2B were purified from rat liver and the rate of exchange of [ ³ H]GDP bound to eIF2 for free, non-radioactively-labeled GDP was measured. The activity of eIF2B was calculated as the slope of the nearest fit line with DPM as the dependent variable and time as the independent variable. [00238] Co-Immunoprecipitation.

[00239] For in vitro co-immunoprecipitations, Sf9 insect cells (1 x 10 ⁶ cells/ml) were infected with recombinant baculovirus encoding FLAG-tagged eIF2Bε subunit alone or co-infected with the three recombinant baculovirus stocks encoding all five eIF2B subunits. At 72 hr post-infection, cells were washed once with ice-cold PBS and lysed with IP buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100 and 10% Glycerol). Cellular debris was removed by centrifugation at 14 00Og for 10 min at 4°C, and the lysates were precleared by mixing with 10 μg of RGS protein and Protein G beads for Ih at 4°C and then centrifuged. The precleared lysate mixture was incubated with anti-Flag M2 monoclonal antibody (2 μg) for 3h at 4°C. The Protein G beads were washed three times with IP buffer and resuspended in 2x Laemmli sample buffer.

[00240] For co-immunoprecipitation of endogenous proteins, confluent UMR- 106 cells in a T75 tissue culture flask were incubated for 3 hr at 37°C with vehicle control or forskolin (100 μM) to upregulate RGS2 expression. Cells were then washed twice with ice-cold PBS and lysed by incubating with ice-cold hypotonic buffer (20 mM Hepes. pH 7.5, 50 mM KCL 0.1 mM PMSF, 1 μg/ml aprotinin, 10 μg/ml leupeptin, 0.1 mM EDTA,

10 % (v/v) glycerol, 1 % Triton X-100) for 10 min followed by repeated aspiration through a 21 gauge needle. Cellular lysates were then centrifuged at 10 00Og and the supernatant was removed and precleared by rotating with 1 μg of mouse IgG and 20 μl of resuspended protein A/G PLUS-agarose for 30 min at 4°C. The precleared cellular

extract (300 μg total cellular protein) was then rotated with anti-eIF2Bε (2 μg) for 1 hr at 4 ⁰ C, and 20 μl of protein A/G PLUS-agarose was subsequently added for an overnight incubation. Immunocomplexes were then washed three times with PBS and resuspended in 2x Laemmli sample buffer. [00241] Immunoblot analysis

[00242] Samples were boiled for 5 min and then run on a 12 % SDS- polyacrylamide gel, transferred to polyvinylidene fluoride membrane, and probed with the appropriate antibodies according to the manufacturer's recommendations. Blots were visualized by LumiGLO Reserve Chemiluminescence (KPL, Inc., Gaithersburg, MD). [00243] Example 2: Nucleic Acids that Encode Peptides that Modulate mRNA

Translation [00244] Results

[00245] H9c2 cells infected with adenovirus coding for full-length RGS2, the

RGS2 minigene, and the eIF2β minigene all exhibited decreased de novo protein synthesis (FIGURE 9) compared to cells infected with a control adenovirus coding for green fluorescent protein (GFP). The decrease in protein synthesis was observed as early as 1 hour and continued for the full 4 hour time-course. The magnitude of the inhibition of cellular protein synthesis is RGS2 minigene > RGS2 = eIF2β minigene. The raw data were expressed as [ ³ H] cpm/μg protein and these values were then normalized as a percentage of the non-specific signal observed at time = 0, which was set to 100%. The data are expressed as mean ± the standard error mean (SEM) of 5 independent experiments performed in triplicate. [00246] Methods

[00247] H9c2 cardiomyoblast cells (American Type Culture Collection, Manassas, VA) were seeded in 12-well cluster plates at a density of 500,000 cells/well and grown to -90% confluency (48 hrs). Cells were then infected with the indicated adenoviruses (GFP [control] or hexahistidine-tagged RGS2 wild type, RGS2 37 amino acid minigene, or eIF2β 37 amino acid "RGS2 homology domain" minigene) at a multiplicity of infection (MOI) of 4. At 48 hours post-infection, the cellular medium was removed and

replaced with 1 ml of Opti-MEM I reduced serum medium (Invitrogen Canada, Burlington, ON) supplemented with 0.5 μCi/ml [ ³ H]-leucine for the indicated times. Cells were then washed with phosphate buffered saline and incubated on ice with ice- cold lysis buffer (20 mM Hepes, pH 7.5, 50 mM KCl, 0.1 mM PMSF, 1 μg/ml aprotinin, 10 μg/ml leupeptin, 0.1 mM EDTA, 10 % (v/v) glycerol, 1 % Triton X- 100) for 15 minutes. The lysate mixture was then transferred to a microcentrifuge tube and spun at maximum speed on a table top centrifuge to pellet the detergent-insoluble membrane fraction. The supernatant was retained for analysis. [00248] Protein concentrations were measured using a Bio-Rad BSA protein assay. An equal volume of the cell lysate was then combined with a 25 % (w/v) solution of trichloroacetic acid to precipitate the protein, subjected to a vigorous vortex, and incubated on ice for 30 minutes. The mixture was filtered through a Millipore filtration apparatus onto glass microfiber disks (GF/C, Whatman, Clifton, NJ) and washed three times each with 5 % TCA followed by 100 % ethanol. Disks were air dried overnight and counted on a Packard Tri-Carb 2900TR liquid scintillation counter (Perkin Elmer,

Boston, MA).

[00249] Prophetic Example 3: Identification of RGS2 and corresponding eIF2Bε-binding domain of eIF2βmolecular determinants of their inhibitory effect on translation

[00250] We will use functional assays to systematically hone in on the amino acid residues in RGS2 that are responsible for its inhibitory effect on translation. As well, we will investigate key amino acid residues in the corresponding eIF2Bε-binding domain of eIF2β to gain further insight and to develop maximally potent and efficacious inhibitory peptides.

[00251] Interestingly, the region of RGS2 identified as the putative eIF2Bε- interacting site displays much similarity to the region of the eIF2β subunit of the eIF2 trimer that binds to eIF2Bε, with 24% identity plus 16% conservative substitutions for an overall similarity of 40% (FIGURE 8). A sequence comparison among all 20 RGS

proteins revealed that this high degree of similarity is unique to RGS2 in spite of the overall similarities shared among RGS proteins in this region. This suggests that eIF2 and RGS2 (but not other RGS proteins) compete for a common binding site on eIF2B. [00252] Using as a reference point this 37 amino acid residue subregion of RGS2, we propose to make and test increasingly shorter truncated forms to eliminate amino acids not required for an effect on translation, as well as analogous RGS2 internal deletion mutants lacking these amino acid residues (e.g., protein RGS2-δ79-116), and finally, point mutations in RGS2 based on similarities and differences between the RGS2 and eIF2β domains illustrated in FIGURE 8. [00253] Truncation mutants

[00254] Since all of the RGS2 truncation mutants that we have made so far have retained their anti-translational activity, it follows that the 37 amino acid stretch identified may exceed the bare minimum required for activity. Therefore we will continue to make larger C- and N-terminal truncations. We have found that as smaller and smaller histidine-tagged RGS2 constucts are made, purification becomes more difficult due to limited protein solubility. If this problem becomes intractable, we will instead generate analogous GST-fusion proteins (which are generally easier to purify) and/or have the corresponding peptides made. [00255] Internal deletions [00256] In addition, we will take a complementary approach wherein amino acid residues that appear to confer inhibitory activity are removed from RGS2. This work has already begun. RGS2-δ79-116 failed to inhibit in vitro translation. We will make smaller internal deletions in this region to identify/confirm the minimal functional domain for this activity. [00257] Substitution mutants

[00258] The 9 identical residues between RGS2 and eIF2β (FIGURE 8) will be replaced in RGS2 by alanine residues, both singly and in groups. If this has has no effect on activity we will repeat the exercise with the 6 conserved residues. It is expected that these amino acid substitutions will produce mutant forms ofRGS2 in which the ability to

block translation is lost or greatly decreased, which thus will point to key eIF2Bε contact points in RGS2. We will also introduce substitutions into RGS2 based on discrepancies with eIF2β, which may increase the affinity of RGS2 for eIF2B. Overall the C-terminal halves of these regions of RGS2 and eIF2β are more similar than the N-terminal halves, where there are four highly nonconservative substitutions between the two proteins (marked by arrows in FIGURE 8), two of which are in the middle of a stretch of 7 dissimilar residues. We will replace these with their counterparts in eIF2β, with the ultimate objective of using this information to produce peptides with improved potency as anti-eIF2 agents. [00259] We will design novel peptides based on our findings. As above, we will examine the effects of these peptides on in vitro translation and we will also test their effects on eIF2B GEF activity (presumably none will have effects on G protein GTPase activity, but we will verify this as well for at least some peptides). Initially, we will design and purchase custom-made peptides corresponding to the putative eIF2Bε- interacting domains of RGS2 and eIF2β (plus flanking residues), and test their abilities to inhibit in vitro translation. Based on the results obtained with these peptides and with our RGS2 mutants, we will design new, improved peptides in an iterative manner, and lest the activities of these in in vitro translation and eIF2B GEF assays. Ultimately, the most active peptides will be tested for their effects on protein production in cells. So far we have been able to test two of these peptides: one which corresponds to the eIF2Bε- interacting domain of eIF2β, and the other being scrambled control peptide based on the most common amino acid residues found in the putative eIF2Bε-interacting domains of both RGS2 and eIF2β. Notably, the eIF 2 β-based peptide caused an essentially complete inhibition of in vitro translation, whereas the control peptide had little or no effect. This experiment clearly shows the feasibility of the proposed approach and provides encouragement that we will be able to optimize this system to generate more potent/efficacious peptides, which may ultimately lead to the development of novel therapeutic agents to inhibit translation.

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[00260] The contents of all cited references including literature references, issued patents, published or non-published patent applications cited throughout this application as well as those listed below are hereby expressly incorporated by reference in their entireties. In case of conflict, the present application, including any definitions herein, will control.

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EQUIVALENTS

[00264] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations. Such equivalents are intended to be encompassed by the following claims.