PEPTIDES AND METHODS OF USE

GOVERNMENT FUNDING

The present invention was made with government support under Grant No. DC02053 and AA-08769, awarded by the National Institutes of Health, Grant No. DAMD17-01- 1-041 1, awarded by the U.S. Army, and Grant No. AQ- 0726, awarded by the Welch Foundation. The Government may have certain rights in this invention.

BACKGROUND

Microtubules are intracellular, filamentous, polymeric structures present in eukaryotic cells that extend throughout the cytoplasm and govern the location of membrane -bounded organelles and other cell components including the chromosomes during cell division. Microtubules are involved in many cellular functions including chromosome migration during mitosis, organelle transport, cytokinesis, cell plate formation, cell motility, and maintenance of cell shape. Microtubules are composed of molecules of tubulin protein, each molecule of which is a heterodimer of α-tubulin and β-tubulin.

Mitosis is the process by which eukaryotic cells ensure the distribution of their chromosomes into two daughter cells during cell division. During this process, cytoplasmic .microtubules are disrupted and reformed as a (mitotic) spindle consisting of large numbers of short microtubules that surround each centrosome. As mitosis proceeds, the elongating ends of the microtubules attach to the chromosomes, the chromosomes align on the metaphase plate, and, during anaphase, the sister chromatids are separated. If any of these stages of chromosomal alignment and separation are disrupted by irregular microtubules, mitosis fails.

Microtubules are the main structures of cellular functions, such as the spatial distribution of organelles and cell motility. In addition, they constitute the scaffold within cilia and flagella. Microtubules are formed by protofilaments of αβ tubulin heterodimers stacked head to tail at 8 nm intervals (Meurer-Grob,

P et al., 2001 , Biochemistry, 40:8000-8008). The sequences of β tubulin isotypes are highly conserved in evolution and even isotypic differences are conserved (Luduena, RF, 1998, Int. Rev. Cytol., 178:207-275). Their main differences lie in the C-terminal structure after helix H 12 composed of 18 amino acid residues. Based on experiments with the testis-specific Drosophila β2 tubulin isotype, it has been suggested that a specific conserved sequence in the C-terminus (termed 'the axonemal motif) plays an important role in axoneme morphogenesis (Nielsen, MG et al., 2001, Curr. Biol., 11 :529-533). In mammals, this sequence is found in the C-terminus of βlV tubulin at positions 433-439, but axonemal motif-like sequences are also found in βl, βll and βV tubulin in the same positions (see Table 1 ).

Table 1. Relationship between the β tubulin C-terminal sequence, the axonemal motif peptide, and isotype-specific βtubulin C-terminal tail peptides, by residue position.

βl, βll, and βV) Notice that the axonemal motif and motif-like sequences pivot around F at position 436 βlll tubulin is 449 amino acids long, and the C-terminal tail peptide is 443SESQGPK (SEQ ID NO 28) AA, amino acid

In recent studies, βl and βV tubulin were found in all mammalian axonemal structures tested, including the cilia of vestibular hair cells, olfactory sensory neurons and ciliated epithelial cells of the nose, trachea, ependyma, fallopian tube and testis (Jensen-Smith, HC et al., 2003, Cell Motil. Cytoskeleton, 55:213-220; Perry, B et al., 2003, J. Assoc. Res. Otolaryngol., 4:329-338; Renthal, R et al., 1993, Cell Motil. Cytoskeleton, 25: 19-29; Woo, K et al., 2002, Cell Tissue Res., 309:331-335). In olfactory neurons, other isotypes (βll and βlll tubulin) were also present (Woo, K et al., 2002, Cell Tissue Res., 309:331-335). Recently, βV tubulin has been found sporadically in axonemal

structures, but is apparently not required for ciliary function and assembly. The only isotypes common to all cilia appear to be βl and βlV tubulin, and their C- termini likely play a key role in ciliary function. There are eight known isotypes of β tubulin in mammals. Their presence and composition are tissue-type specific, but no functional correlation has been made to date (Hallworth, R et ah, 2000, Hear. Res., 148: 161-172; Luduena, RF, 1993, MoI. Biol. Cell, 4:445-457).

Recent evidence indicates that the C-terminus of β-tubulin provides a contact surface on microtubules that is utilized by the tubulin motors, kinesin and dynein, to actively move along the microtubule surface in a directional manner (Case, RB et ah, 1997, Cell, 90:959-966; Okada, Y et ah, 2003, Nature, 424:574-577). Kinesin or dynein motors play an active role in cell division, viral infection and maturation, bacterial and parasite infection. These tubulin motors play a key role in cell division and so are potential targets for treating cancer or other specific diseases such as arthritis where the disease features the pathological proliferation of certain cell types. Proliferation of synovial cells is considered to play a key role in rheumatoid arthritis. Taxol, a tubulin inhibitor, has been shown in animals to cause the regression of a collagen induced arthritis, a model of rheumatoid arthritis (Brahn, E et ah, 1994, Arthritis Rheum., 37:839- 845; Houri, JM et ah, 1995, Curr. Opin. Rheumatol., 7:201-205). Kinesin motors play an important role in spindle and chromosome motility. Kinesin motors are necessary for establishing spindle bipolarity, positioning chromosomes on the metaphase plate, and maintaining forces in the spindle. It also appears that kinesin motors can facilitate microtubule depolymerization, raising the possibility that the motors modulate microtubule dynamics during mitosis. The force generated by microtubule polymerization/depolymerization is thought to contribute to, or underlie, spindle dynamics and movements of the chromosomes.

Kinesin proteins that associate with chromosomes along the length of their arms have recently been reported and named Kinesin-4 (formerly Chromokinesin or KIF4) motors. XkIp 1 , a chromokinesin of Xenopus, is required for maintenance of spindle bipolarity and congression of chromosomes to the metaphase plate. Nod, a meiotic kinesin in Drosophila, has been postulated to perform an analogous role in oocyte meiosis of positioning chromosomes on the metaphase plate.

The chromokinesins are a subfamily of kinesins that are associated with the chromosome both as a structural component and a tubulin motor (Mazumdar, M et al., 2005, Trends Cell Biol., 15:349-355). They are clearly crucial components of the mitotic machinery and are required for accurate genome segregation. Not surprisingly, loss of chromokinesin function leads to deleterious genome defects, particularly an increased number of anaphase bridges and micronuclei. The most direct evidence for a role for chromokinesins in genome stability is the observed numerical aneuploidy observed upon depletion of HKXF4A. Indeed, HKIF4 was identified as a potential protooncogene in the transition from normal extravillous trophoblast (EVT) cells to a premalignant stage.

Furthermore, the reported interaction of HKIF4A with BRAF-35, a BRCA2-associated factor, in a yeast two-hybrid assay is interesting in the context of a proposed function of the tumor-suppressor BRCA2 in cytokinesis, allowing for the possibility that BRAF-35/BRCA2 are involved in chromosome condensation or that chromokinesins are linked to the DNA repair machinery by means of BRC A2. However, direct evidence for a role for chromokinesin in tumor formation is lacking. By analogy with several other mitotic regulators, the involvement of chromokinesins in accurate genome segregation might make them suitable targets for anticancer therapeutics. The feasibility of this approach is demonstrated by the identification of a specific smallmolecule inhibitor for the mitotic spindle kinesin Eg5. Human Eg5-specific anti-tumor agents have now been tested for their potency to inhibit tumor growth. Targeting the more specifically acting chromokinesins might provide an improvement over the currently used generally acting anti-microtubule cancer drugs such as taxol or nocodazole (Mandelkow, E et al., 2002, Trends Cell Biol., 12:585-591). A large number of kinesins have been implicated in mitosis. The compound adociasulfate-2 from a marine sponge blocks kinesin-dependent motility and mitosis and is an example of such a drug. Another example is the small molecular weight inhibitor 'monastrol' (Kapoor, TM et al., 2000, J. Cell Biol., 150:975-988). Dynein also plays a key role in the formation of poles and capture of chromosomes in spindle assembly.

There remains a need in the art for effective agents for use in cancer therapy and other cell proliferative disorders and/or disorders in which tubulin motor proteins activity plays a role in the progression of the disorder.

SUMMARY OF THE INVENTION

The invention is directed to isolated peptides (i.e., polypeptides) having (preferably consisting essentially of, and more preferably consisting of) the sequence EGEFXXX, where X is either glutamic (E) or aspartic (D) acid (SEQ ID NO:1) or its active analogs. The active analogs (and preferably the isolated peptides of the invention) bind to at least one member of the class of tubulin motor proteins (kinesin or dynein). More preferably, the active analogs (and preferably the isolated peptides of the invention) inhibit the ability of at least one tubulin motor protein to move on microtubules. The peptide EGEFXXX, where X is either glutamic (E) or aspartic (D) acid, (SEQ ID NO: 1) is based on the C-terminus of the human β tubulin. A shorter or longer form of this peptide sequence may be active. Thus, in one embodiment, the present invention provides an isolated polypeptide having no more than 33 amino acids and having the sequence EGEFXXX, wherein X is either glutamic (E) or aspartic (D) acid (SEQ ID NO:1), or an active analog thereof, wherein the active analog binds to at least one member of the class of tubulin motor proteins and includes 6 to 33 amino acids.

Preferably, the isolated polypeptides of the invention are derived from the C-terminus of a β-tubulin isoform. Thus, in one embodiment, the present invention provides an isolated polypeptide derived from the C-terminus of a β- tubulin isoform, wherein the isolated polypeptide binds to at least one member of the class of tubulin motor proteins.

The present invention provides a method of selectively blocking motor protein interaction with tubulin, preferably β-tubulin. The method includes introducing into a cell a polypeptide of the present invention.

The present invention provides a method for treating (e.g., preventing and/or reversing) a cell proliferative disorder (e.g., tumorogenesis, arthritis) in a subject. The method includes administering to the subject an effective amount

of a composition including a polypeptide described herein. In one embodiment, the present invention provides a method of treating (e.g., preventing and/or reversing) tumorigenesis in a subject. The method includes administering to the subject an effective amount of a composition including a polypeptide described herein.

This invention provides an active agent for treating disorders (e.g., diseases) where tubulin motor protein activity plays a role in the progression of the disorder (e.g., bacterial infection, a protozoal infection, and/or a viral infection). The method includes administering to the subject an effective amount of a composition including a polypeptide described herein.

These peptidic inhibitors can be used in vitro or in vivo, including internal and external use in animals, including mammals such as humans. They can be used for preventative (i.e., prophylactic) treatments or for therapeutic treatments.

Definitions

Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one. Thus, a composition that includes "a" polypeptide refers to a composition that includes one or more polypeptides. Herein, a "composition" could include just the peptide.

The terms "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims.

Room temperature, as defined herein, is the ambient temperature that a room used for human habitation is generally maintained at, and is generally a temperature from 20 ⁰ C to 25 ⁰ C, with 22.5°C being particularly preferred.

The terms "polypeptide" and "peptide" are used interchangeably herein to refer to a polymer of amino acids. These terms do not connote a specific length of a polymer of amino acids. Thus, for example, the terms oligopeptide, protein, and enzyme are included within the definition of polypeptide or peptide, whether produced using recombinant techniques, chemical or enzymatic synthesis, or naturally occurring. This term also includes polypeptides that have been modified or derivatized, such as by glycosylation, acetylation, phosphorylation, and the like.

"Amino acid" is used herein to refer to a chemical compound with the general formula: NH ₂ -CRH-COOH, where R, the side chain, is H or an organic group. Where R is organic, R can vary and is either polar or nonpolar (i.e., hydrophobic). The amino acids of this invention can be naturally occurring or synthetic (often referred to as nonproteinogenic). As used herein, an organic group is a hydrocarbon group that is classified as an aliphatic group, a cyclic group or combination of aliphatic and cyclic groups. The term "aliphatic group" means a saturated or unsaturated linear or branched hydrocarbon group. This term is used to encompass alkyl, alkenyl, and alkynyl groups, for example. The term "cyclic group" means a closed ring hydrocarbon group that is classified as an alicyclic group, aromatic group, or heterocyclic group. The term "alicyclic group" means a cyclic hydrocarbon group having properties resembling those of aliphatic groups. The term "aromatic group" refers to mono- or polycyclic aromatic hydrocarbon groups. As used herein, an organic group can be substituted or unsubstituted.

The following abbreviations are used throughout the application: A = Ala = Alanine, T = Thr = Threonine, V = VaI = Valine, C = Cys = Cysteine, L = Leu = Leucine, Y = Tyr = Tyrosine, I = He = Isoleucine, N = Asn = Asparagine, P = Pro = Proline, Q = GIn = Glutamine, F = Phe = Phenylalanine, D = Asp = Aspartic Acid, W = Tip = Tryptophan, E = GIu = Glutamic Acid, M = Met =

Methionine, K = Lys = Lysine, G = GIy = Glycine, R = Arg = Arginine, S = Ser = Serine, H = His = Histidine.

Herein, the term "tubulin" refers to the family of β tubulin proteins. Thus, this term encompasses βi, βn, βm. βrv, and β _v tubulin. The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 provides graphs showing (A) the effect of isotype specific antibodies against β tubulin on cilary beat frequency CBF. Final antibody concentrations 0.33 μM, error bars indicate 1 s.e.m. Solid squares, control; open circles, anti βl tubulin; open upward triangles, anti βll tubulin; open downward triangles, anti βlll tubulin; open diamonds, anti βFV tubulin; open triangles, anti βV tubulin. The antibodies depicted in circles or diamonds blocked ciliary beating, the antibodies in upward traingles had no effect on CBF; (B) the concentration dependence of anti-βl tubulin; error bars indicate 1 s.e.m. Antibody concentrations: upward triangles in panel A, 0.33 μM; upward triangles in panel B, C, D, 0.033 μM; circles, 3.3 nM. Similar concentration curves were obtained for anti-βlV tubulin and anti-βV tubulin; and (C) the effect of non-isotype-specific, non-C-terminal antibodies against α and β tubulin (DMlA, circle and TUB 2.1, upward triangles panel B, C, D), error bars indicate 1 s.e.m.; antibody concentrations 0.33 μM. (D) Effect of anti-β tubulin C- (circle) and N-terminal (upward triangles, panel B, C, D), nonisotype- specific antibodies, error bars indicate 1 s.e.m.; antibody concentrations 0.33 μM.

Figure 2 provides graphs providing a comparison of the effect of axonemal motif (open triangles) and C-terminal tail peptides (open circles) on CBF and controls (dotted line, solid squares) error bars indicate 1 s.e.m.; peptide concentrations: 0.33 μM. For sequences, see Table 1. Figure 2(A) sequences derived from βl tubulin (EEDFGEE (SEQ ID NO:7) and EEAEEEA (SEQ ID NO:8)); figure 2(B) sequences derived from βll tubulin (QGEFEEE (SEQ ID NO:9) and EGEDEA (SEQ ID NO: 10)); figure 2(C) sequences derived from βlll tubulin (GEMYEDD (SEQ ID NO:11) and SESQGPK (SEQ ID NO: 12)); figure 2(D) sequences derived from βlV tubulin (EGEFEEE (SEQ ID NO: 13) and EAEEEVA (SEQ ID NO: 14)); figure 2(E) sequences derived from βV tubulin (EEAFEDE (SEQ ID NO: 15) and EEEINE (SEQ ID NO: 16)); and figure 2(F) concentration-dependent effect of the axonemal motif peptide (EGEFEEE (SEQ ID NO: 17)), concentrations: 0.33 μM (squares), 0.033 μM (circles) and 3.3 nM (triangles).

Figure 3 provides graphs showing (A) an alanine scan of the axonemal motif peptide: when the central F is replaced by an A, there is no more effect of

the peptide on CBF, circles, EGEFEEE (SEQ ID NO: 17), triangles, EGEAEEE (SEQ ID NO: 18). Peptide concentrations: 0.33 μM. Error bars indicate 1 s.e.m; and (B) the effect of β tubulin axonemal motif-like peptides on CBF, circles, EGEGEEE (SEQ ID NO: 19), triangles, EDEDEGE (SEQ ID NO:20). Peptide concentrations: 0.33 μM. Error bars indicate 1 s.e.m.

Figure 4 provides pictures showing (A) an example of a labeling of isolated bovine tracheal cilia by the isotype-specific antibody against βlV tubulin (scale bar 10 μm) and (B) immunoblotting of isolated bovine tracheal cilia by isotype-specific antibodies against α and β tubulin.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS OF THE

INVENTION

The present invention is directed to isolated polypeptides that have the sequence EGEFXXX, wherein X is either glutamic (E) or aspartic (D) acid (SEQ ID NO: 1), or active analogs thereof. Preferably, certain of these peptides are isolated from the C-terminus of the human β-tubulin.

The active analogs (and preferably the isolated peptides of the invention) bind to at least one member of the class of tubulin motor proteins (kinesin or dynein). More preferably, the active analogs (and preferably the isolated peptides of the invention) inhibit the ability of at least one tubulin motor protein to move on microtubules.

The function of the tubulin motor proteins is to move client proteins or cargoes (such as vesicles or chromosomes) along microtubules so that they can perform specific functions in specific locations or separate cargoes including chromosomes in living cells. This discovery of a peptide that binds to at least one member of the class of tubulin motor proteins (preferably, in a very specific fashion) makes it a candidate inhibitor of microtubule function, and provides a novel class of inhibitors that can be used as a new front to defeat cancer, for example. The polypeptides of this invention can be added to cells in culture or used to treat subjects, such as mammals. Where the polypeptides are used to treat a subject, the polypeptides are preferably combined in a pharmaceutical composition with a pharmaceutically acceptable carrier, such as a larger

molecule to promote polypeptide stability or a pharmaceutically acceptable buffer that serves as a carrier for the polypeptide.

Treatment can be prophylactic or therapeutic. Thus, treatment can be initiated before, during, or after the development of the condition (e.g., cancer). As such, the phrases "treating," "inhibiting," "effective to inhibit," and the like, include both prophylactic and therapeutic treatment (i.e., prevention and/or reversal of the condition).

The peptides of the invention include the sequence EGEFXXX, where X is either glutamic (E) or aspartic (D) acid (SEQ ID NO:1), or active analogs thereof. Peptides of the invention can be shorter (e.g., as short as 6 amino acids) or longer (e.g., up to 24 amino acids, and preferably up to 18 amino acids) than the sequence EGEFXXX, where X is either glutamic (E) or aspartic (D) acid (SEQ TD NO: 1 ). Preferably, however, peptides of the invention are no shorter than 7 amino acids. As used in this context, an "active analog" of EGEFXXX, where X is either glutamic (E) or aspartic (D) acid (SEQ ID NO: 1) binds to at least one member of the tubulin motor class of proteins and preferably alters its ability to move along a tubulin microfilament.

Peptides of the invention, whether they include the sequence EGEFXXX, where X is either glutamic (E) or aspartic (D) acid (SEQ ID NO:1) or its active analogs, preferably bind to at least one member of the tubulin motor class of proteins (e.g., kinesin or dynein), and more preferably to kinesins involved in mitosis. Preferably, peptides of the invention bind to the tubulin motor protein blocking the ability of that protein to move along a tubulin filament. In one embodiment of the invention, isolated polypeptides including the sequence EGEFXXX, where X is either glutamic (E) or aspartic (D) acid (SEQ ID NO: 1) or its active analogs, are used in a method of selectively blocking tubulin motor protein interaction with tubulin. While not intending to be bound by theory, the isolated polypeptides preferably selectively block tubulin motor protein interaction with tubulin by occupying or blocking at least a portion of the tubulin binding site on the tubulin motor protein. Blocking of the tubulin binding site of the tubulin motor protein preferably decreases binding of the tubulin motor protein to tubulin, thereby decreasing the ability of the motor protein to move along a tubulin filament.

A list of kinesin sequences and their respective family classification is actively maintained online on the World Wide Web by the proWeb project under "Kinesin Proteins listed by Organism" at, for example, http://www.proweb.Org/kinesin//MotorSeqTable.html. A list of dynein heavy chains is given in Table 2.

Table 2. Dynein heavy chains, sequence ID, and chromosomal location.

Active analogs of EGEFXXX, where X is either glutamic (E) or aspartic

(D) acid (SEQ ID NO: 1), include polypeptides having structural similarity to

those of SEQ ID NO: 1. Preferably, an active analog is a polypeptides with an amino acid sequence that shares at least 80% amino acid identity to any polypeptide of SEQ ID NO: 1 ; more preferably, it shares at least 90% amino acid identity therewith, most preferably, at least 95% amino acid identity. Amino acid identity is defined in the context of a comparison between an active analog of EGEFXXX (SEQ ID NO: 1) and any polypeptide of SEQ ID NO: 1, and is determined by aligning the residues of the two amino acid sequences to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical amino acids, although the amino acids in each sequence must nonetheless remain in their proper order. Preferably, two amino acid sequences are compared using the Blastp program, version 2.2.10, of the BLAST 2 search algorithm, as described by Tatusova (Tatusova, TA et al., 1999, FEMS Microbiol. Lett., 174:247-250), and available on the world wide web at the National Center for Biotechnology Information website, under BLAST in the Molecular Database section. Preferably, the default values for all BLAST 2 search parameters are used, including matrix = BLOSUM62; open gap penalty = 1 1, extension gap penalty = 1 , gap x_dropoff = 50, expect = 10, wordsize = 3, and optionally, filter on. In the comparison of two amino acid sequences using the BLAST search algorithm, structural similarity is referred to as "identity."

Preferably, polypeptides of the invention (including active analogs) can be as small as 6 amino acids or as long as 33 amino acids (more preferably as long as 24 amino acids, even more preferably as long as 18 amino acids, and even more preferably as long as 7 amino acids). Active analogs could include specific modifications that would confer the appropriate stability to the peptide, would confer the ability of the peptide to be properly localized in specific tissue or in a specific compartment of the cell. Thus, the peptide may be modified (e.g., chemically) so that it will be taken up by target cells more readily. For example, it may be constructed in a divalent or multivalent form. Active analogs could include specific post translational modifications such as acetylation, phosphorylation, polyglutamylation, polyglycylation and detyrosination or the incorporation of specific amino acid analogs that would stabilize the peptide in a pharmaceutically active conformation.

Active analogs can have conservative amino acid substitutions. Such active analogs are referred to as "conservatively modified variants." For the purposes of this invention, conservative amino acid substitutions are defined to result from exchange of amino acids residues from within one of the following classes of residues: Class I: Ala, GIy, Ser, Thr, and Pro (representing small aliphatic side chains and hydroxyl group side chains); Class II: Cys, Ser, Thr and Tyr (representing side chains including an —OH or -SH group); Class III: GIu, Asp, Asn and GIn (carboxyl group containing side chains):Class IV: His, Arg and Lys (representing basic side chains); Class V: lie, VaI, Leu, Phe and Met (representing hydrophobic side chains); and Class VI: Phe, Trp, Tyr and His

(representing aromatic side chains). The classes also include related amino acids such as 3Hyp and 4Hyp in Class I; homocysteine in Class II; 2-aminoadipic acid, 2-aminopimelic acid, gamma-carboxyglutamic acid, beta-carboxyaspartic acid, and the corresponding amino acid amides in Class III; ornithine, homoarginine, N-methyl lysine, dimethyl lysine, trimethyl lysine, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, homoarginine, sarcosine and hydroxylysine in Class IV; substituted phenylalanines, norleucine, norvaline, 2-aminooctanoic acid, 2- aminoheptanoic acid, statine and beta-valine in Class V; and naphthylalanines, substituted phenylalanines, tetrahydroisoquinoline-3-carboxylic acid, and halogenated tyrosines in Class VI.

Preferably, active analogs are derived from the C-terminus of a tubulin isoform. Examples of such active analogs include:

Active analogues can include any beta tubulin isoform C-terminus sequence including the isolated peptides that have the sequence,

LVSEYQQYQDATAEEEGEMYEDDEEESEAQGPK (SEQ ID NO:29) LVSEYQQYQDATAEEEEDFGEEAEEEA (SEQ ID NO:30), LVSEYQQYQDATAEEEEDFGEEAEEEA (SEQ ID NO: 31), LVSEYQQYQDATAEEEGEFEEEAEEEVA (SEQ ID NO: 32), LVSEYQQYQDATAEQGEFEEEAEEEVA ( SEQ ID NO:33), or their active analogs.

EGEFXXX where X is either glutamic (E) or aspartic (D) acid (SEQ ID NO: 1), or any combination of its peptide fragments or other active analogs

thereof, for example, can be synthetically constructed using known peptide polymerization techniques, can be isolated from a natural source, or can be produced using recombinant techniques, or chemically or enzymatically synthesized. For example, the peptides of the invention may be synthesized by the solid phase method using standard methods based on either t-butyloxycarbonyl (BOC) or 9-fluorenylmethoxy-carbonyl (FMOC) protecting groups. This methodology is described by G. B. Fields et al. in Synthetic Peptides: A User's Guide, W. M. Freeman & Company, New York, N.Y., pp. 77-183 (1992). Moreover, gene sequence encoding the EGEFXXX, where X is either glutamic (E) or aspartic (D) acid (SEQ ID NO: 1) peptide or analogs thereof can be constructed by known techniques such as expression vectors or plasmids and transfected into suitable microorganisms that will express the DNA sequences thus preparing the peptide for later extraction from the medium in which the microorganism are grown. For example, U.S. Pat. No. 5,595,887 describes methods of forming a variety of relatively small peptides through expression of a recombinant gene construct coding for a fusion protein that includes a binding protein and one or more copies of the desired target peptide. After expression, the fusion protein is isolated and cleaved using chemical and/or enzymatic methods to produce the desired target peptide.

Use of Polypeptides of the Invention

Cell proliferative disorders include, but are not limited to, arthritis, tumorogenesis (including benign tumors), blood vessel proliferative disorders, autoimmune disorders, and/or fibrotic disorders. Specific examples include atherosclerosis, restenosis, retinopathy, retinoblastoma, osteosarcoma, breast cancer, bladder cancer, prostate cancer, renal carcinoma, cancers associated with viral infections, such as cervical cancers associated with human papilloma virus, and small-cell lung cancer. Additionally, the methods of the invention are useful for the treatment of one or more infections that depend on tubulin motors for part of their infective cycle. Such infections include, for example, bacterial infections (Escalante- Ochoa, C et al., 2000, Microb. Pathog., 28:321-333), protozoal infections (Armson, A et al., 2003, Expert. Rev. Anti. Infect. Ther., 1 :297-305; Werbovetz,

KA, 2002, Mini. Rev. Med. Chem., 2:519-529), and viral infections (Mandelkow, E et al., 2002, Trends Cell Biol., 12:585-591), including retroviral infections. Viruses known to depend on microtubules during their infective cycle includes adenovirus (Leopold, PL et al., 2000, Hum. Gene Then, 1 1 : 151- 165), retrovus (Kim, W et al., 1998, J. Virol., 72:6898-6901) such as HlV (Battaglia, PA et al., 2001, J. Cell Sci., 114:2787-2794), and Herpes simplex (Diefenbach, RJ et al., 2002, J. Virol., 76:3282-3291).

The preferred effect of the methods of the instant invention, with respect to cell proliferative disorders, is the inhibition, to some extent, of growth of cells causing or contributing to a cell proliferative disorder. A therapeutic effect relieves to some extent one or more of the symptoms of a cell proliferative disorder. In reference to the treatment of a cancer, a therapeutic effect refers to one or more of the following: 1) reduction in the number of cancer cells; 2) reduction in tumor size; 3) inhibition (i.e., slowing to some extent, preferably stopping) of cancer cell infiltration into peripheral organs; 4) inhibition (i.e., slowing to some extent, preferably stopping) of tumor metastasis; 5) inhibition, to some extent, of tumor growth; and/or 6) relieving to some extent one or more of the symptoms associated with the disorder.

In reference to the treatment of a cell proliferative disorder other than a cancer, a desired effect refers to either: 1) the inhibition, to some extent, of the growth of cells causing the disorder; 2) the inhibition, to some extent, of the production of factors (e.g., growth factors) causing the disorder; and/or 3) relieving to some extent one or more of the symptoms associated with the disorder. Cell proliferative disorders, including those referenced above, are not necessarily independent. For example, fϊbrotic disorders may be related to, or overlap with, blood vessel disorders. Additionally, for example, atherosclerosis (which is characterized herein as a blood vessel disorder) is associated with the abnormal formation of fibrous tissue. A cancer cell refers to various types of malignant neoplasms, most of which can invade surrounding tissues, and may metastasize to different sites. The formation and spreading of blood vessels; i.e., vasculogenesis and angiogenesis, respectively, play important roles in a variety of physiological processes such as embryonic development, wound healing and organ

regeneration. They also play a role in cancer development. Blood vessel proliferation disorders refer to angiogenic and vasculogenic disorders generally resulting in abnormal proliferation of blood vessels. Examples of such disorders include restenosis, retinopathies, and atherosclerosis. As noted above, other such proliferative disorders can be identified by standard techniques, and by determination of the efficacy of action of the compounds described herein.

Formulation and Administration of Polypeptides of the Invention The present invention also provides a composition that includes one or more active agents (e.g., EGEFXXX, where X is either glutamic (E) or aspartic (D) acid (SEQ ID NO: 1), at least one active analog thereof) of the invention, and optionally one or more carriers, preferably a pharmaceutically acceptable carrier, which could be a pharmaceutically acceptable buffer. The peptides of the present invention may be employed in a monovalent state (i.e., free peptide or a single peptide fragment coupled to a carrier molecule). The peptides may also be employed as conjugates having more than one (same or different) peptide fragment bound to a single carrier molecule. The carrier may be a biological carrier molecule (e.g., a glycosaminoglycan, a proteoglycan, albumin or the like) or a synthetic polymer (e.g., a polyalkyleneglycol or a synthetic chromatography support). Typically, ovalbumin, human serum albumin, other proteins, polyethylene glycol, or the like, are employed as the carrier. Such modifications may increase the apparent affinity and/or change the stability of a peptide. The number of peptide fragments associated with or bound to each carrier can vary, but from about 4 to 8 peptides per carrier molecule are typically obtained under standard coupling conditions.

For instance, peptide/carrier molecule conjugates may be prepared by treating a mixture of peptides and carrier molecules with a coupling agent, such as a carbodiimide. The coupling agent may activate a carboxyl group on either the peptide or the carrier molecule so that the carboxyl group can react with a nucleophile (e.g., an amino or hydroxyl group) on the other member of the peptide/carrier molecule, resulting in the covalent linkage of the peptide and the carrier molecule. For example, conjugates of a peptide coupled to ovalbumin

may be prepared by dissolving equal amounts of lyophilized peptide and ovalbumin in a small volume of water. In a second tube, l-ethyl-3-(3- dimethylamino-propy])-carbodiimide hydrochloride (EDC; ten times the amount of peptide) is dissolved in a small amount of water. The EDC solution is added . to the peptide/ovalbumin mixture and allowed to react for a number of hours. The mixture may then be dialyzed (e.g., into phosphate buffered saline) to obtain a purified solution of peptide/ovalbumin conjugate. Peptide/carrier molecule conjugates prepared by this method typically contain about 4 to 5 peptides per ovalbumin molecule. The methods of the invention typically include administering to (e.g., applying to the skin of) a subject (preferably a mammal, and more preferably a human) a composition of the invention in an amount effective to produce the desired effect. The term "effective amount" as used herein, means an amount of a peptide utilized in the methods of the present invention that is capable of providing a desired effect. The specific dose of compound administered according to this invention to obtain therapeutic and/or prophylactic effects will, of course, be determined by the particular circumstances surrounding the case, including, for example, the compound administered, the route of administration, the condition being treated and the individual being treated. Useful dosages of the active agents can be determined by comparing their in vitro activity and the in vivo activity in animal models. Methods for extrapolation of effective dosages in mice, and other animals, to humans are known in the art; for example, see U.S. Pat. No. 4,938,949. A typical daily dose (administered in single or divided doses) will contain a dosage level of from about 0.01 mg/kg to about 50 mg/kg of body weight of a peptide of this invention. Preferred daily doses generally will be from about 0.05 mg/kg to about 20 mg/kg and ideally from about 0.1 mg/kg to about 10 mg/kg.

The active agents of the present invention can be formulated for enteral administration (oral, rectal, etc.) or parenteral administration (injection, internal pump, etc.). The administration can be via direct injection into tissue, intraarterial injection, intravenous injection, or other internal administration procedures, such as through the use of an implanted pump, or via contacting the composition with a mucus membrane in a carrier designed to facilitate transmission of the composition across the mucus membrane such as a

suppository, eye drops, inhaler, or other similar administration method or via oral administration in the form of a syrup, a liquid, a pill, capsule, gel coated tablet, or other similar oral administration method. The active agents can be incorporated into an adhesive plaster, a patch, a gum, and the like, or it can be encapsulated or incorporated into a bio-erodible matrix for controlled release. The carriers for internal administration can be any carriers commonly used to facilitate the internal administration of compositions such as plasma, sterile saline solution, IV solutions, or the like. Carriers for administration through mucus membranes can be any well known in the art. Carriers for administration oral can be any carrier well known in the art.

The formulations may be conveniently presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active agent into association with a carrier that constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulations.

Formulations suitable for parenteral administration conveniently include a sterile aqueous preparation of the active agent, or dispersions of sterile powders of the active agent, which are preferably isotonic with the blood of the recipient. Isotonic agents that can be included in the liquid preparation include sugars, buffers, and sodium chloride. Solutions of the active agent can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions of the active agent can be prepared in water, ethanol, a polyol (such as glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, glycerol esters, and mixtures thereof. The ultimate dosage form is sterile, fluid, and stable under the conditions of manufacture and storage. The necessary fluidity can be achieved, for example, by using liposomes, by employing the appropriate particle size in the case of dispersions, or by using surfactants. Sterilization of a liquid preparation can be achieved by any convenient method that preserves the bioactivity of the active agent, preferably by filter sterilization. Preferred methods for preparing powders include vacuum drying and freeze drying of the sterile injectable solutions. Subsequent microbial contamination can be prevented using various antimicrobial agents, for example, antibacterial,

antiviral and antifungal agents including parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. Absorption of the active agents over a prolonged period can be achieved by including agents for delaying, for example, aluminum monostearate and gelatin. Formulations of the present invention suitable for oral administration may be presented as discrete units such as tablets, troches, capsules, lozenges, wafers, or cachets, each containing a predetermined amount of the active agent as a powder or granules, as liposomes containing the active agent, or as a solution or suspension in an aqueous liquor or non-aqueous liquid such as a syrup, an elixir, an emulsion, or a draught. The amount of active agent is such that the dosage level will be effective to produce the desired result in the subject. Nasal spray formulations include purified aqueous solutions of the active agent with preservative agents and isotonic agents. Such formulations are preferably adjusted to a pH and isotonic state compatible with the nasal mucous membranes. Formulations for rectal or vaginal administration may be presented as a suppository with a suitable carrier such as cocoa butter, or hydrogenated fats or hydrogenated fatty carboxylic acids. Ophthalmic formulations are prepared by a similar method to the nasal spray, except that the pH and isotonic factors are preferably adjusted to match that of the eye. Topical formulations include the active agent dissolved or suspended in one or more media such as mineral oil, DMSO, polyhydroxy alcohols, or other bases used for topical pharmaceutical formulations.

The tablets, troches, pills, capsules, and the like may also contain one or more of the following: a binder such as gum tragacanth, acacia, corn starch or gelatin; an excipient such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; a sweetening agent such as sucrose, fructose, lactose or aspartame; and a natural or artificial flavoring agent. When the unit dosage form is a capsule, it may further contain a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac, or sugar and the like. A syrup or elixir may contain one or more of a sweetening agent, a preservative such as methyl- or propylparaben, an agent to retard crystallization

of the sugar, an agent to increase the solubility of any other ingredient, such as a polyhydric alcohol, for example glycerol or sorbitol, a dye, and flavoring agent. The material used in preparing any unit dosage form is substantially nontoxic in the amounts employed. The active agent may be incorporated into sustained- release preparations and devices.

In certain embodiments of the invention, the active agents of the present invention can be used in cosmetic formulations (e.g., skincare cream, sunscreen, decorative make-up products, and other dermatological compositions) in various pharmaceutical dosage forms, and especially in the form of oil-in-water or water-in-oil emulsions, solutions, gels, or vesicular dispersions. The cosmetic formulations may take the form of a cream that can be applied either to the face or to the scalp and hair, as well as to the human body. They can also serve as a base for a lipstick.

Particularly preferred cosmetic formulations can also include additives such as are usually used in such formulations, for example preservatives, bactericides, perfumes, antifoams, dyes, pigments which have a coloring action, surfactants, thickeners, suspending agents, fillers, moisturizers and/or humectants, fats, oils, waxes or other customary constituents of a cosmetic formulation, such as alcohols, polyols, polymers, foam stabilizers, electrolytes, organic solvents, or silicone derivatives.

Cosmetic formulations typically include a lipid phase and often an aqueous phase. The lipid phase can advantageously be chosen from the following group of substances: mineral oils, mineral waxes oils, such as triglycerides of capric or of caprylic acid, but preferably castor oil; fats, waxes and other natural and synthetic fatty substances, preferably esters of fatty acids with alcohols of low C number, for example with isopropanol, propylene glycol or glycerol, or esters of fatty alcohols with alkanoic acids of low C number or with fatty acids; alkyl benzoates; silicone oils, such as dimethylpolysiloxanes, diethylpolysiloxanes, diphenylpolysiloxanes and mixed forms thereof. If appropriate, the aqueous phase of the formulations according to the invention advantageously includes alcohols, diols or polyols of low C number and ethers thereof, preferably ethanol, isopropanol, propylene glycol, glycerol, ethylene glycol, ethylene glycol monoethyl or monobutyl ether, propylene glycol monomethyl, monoethyl or monobutyl ether, diethylene glycol monomethyl or

monoethyl ether and analogous products, furthermore alcohols of low C number, for example ethanol, isopropanol, 1,2-propanediol, and glycerol, and, in particular, one or more thickeners, which can advantageously be chosen from the group consisting of silicon dioxide, aluminium silicates, polysaccharides and derivatives thereof, for example hyaluronic acid, xanthan gum, and hydroxypropylmethylcellulose, particularly advantageously from the group consisting of poly-acrylates, preferably a polyacrylate from the group consisting of so-called CARBOPOLS, for example CARBOPOLS of types 980, 981 , 1382, 2984, and 5984, in each case individually or in combination.

The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope of the invention as set forth herein.

EXAMPLES

Example 1 To investigate the functional significance of the C-termini of β tubulin isotypes in mammals, a preparation of isolated, de-membranated ATP- activated beating bovine cilia was used (Hastie, AT et al., 1986, Cell Motil. Cytoskeleton, 6:25-34; Wyatt, TA et ah, 2005, Am. J. Physiol Lung Cell MoI. Physiol, 288:L546-L551). The effect of isotype-specific monoclonal antibodies directed against the C-termini of tubulin and monoclonal antibodies directed against other epitopes of β tubulin, as well as monoclonal antibodies against various epitopes of β tubulin, on ciliary beat frequency (CBF) was measured using a new, rapid digital video motility analysis method (Sisson, JH et ah, 2003, J. Microsc, 21 1 : 103-1 1 1). In addition, the effect on CBF of peptides containing (1) the C-terminal amino acid sequences of β tubulin isotypes against which the antibodies were raised (C-terminal tail peptides, CTT peptides) and (2) the peptides containing the amino acid sequences of the isotype-specific axonemal motif or the closest equivalent, was examined.

Preparation of cilia

Isolated bovine tracheal cilia were prepared from fresh bronchi by the method of Hastie et al. (Hastie, AT, 1995, Methods Cell Biol., 47:93-98; Hastie, AT et al., 1986, Cell Motil. Cytoskeleton, 6:25-34). Tracheas from bovine lungs were obtained from a local abattoir, dissected, cleaned from debris and rinsed in cold PBS (pH 7.4). The orifices were clamped and the tracheas were then incubated with approximately 200 milliliters (ml) of a Triton X-100- based cilia extraction buffer under continuous shaking for 90 seconds (following the procedures of Hastie, containing Tris HCl (20.0 millimolar (rnM)), NaCl (50.0 mM), CaCl ₂ (10.0 mM), EDTA (1.0 mM), 2-mercaptoethanol (7.0 mM), Triton X-100 (100.0 mM) and dithiotreitol (DTT, 1.0 mM) pH 7.4)). The fluid containing cilia was filtered through micromesh (pore size 0.45 micrometer (μm)). After centrifugation at 10,000 gravities (g) at 4°C for 7 minutes, the supernatant was discarded and the pelleted cilia were resuspended in resuspension buffer (Tris HCl (20.0 mM), KCl (50.0 mM), MgCl ₂ -OH ₂ O (4.0 mM), EDTA (0.5 mM), DTT (1.0 mM), Soybean Trypsin Inhibitor (10.0 mM), pH 8.0). The protein concentration was determined using a Bradford-dye assay. The cilia preparation was frozen in a 25 wt-% (percent by weight) sucrose solution in a concentration of one milligram(mg)/milliliter(ml) in 100 microliter (μl) aliquots and stored at -8O ⁰ C

Activation of cilia and measuring ciliary beat frequency (CBF)

A thawed aliquot of frozen demembranated cilia was mixed with resuspension buffer and cAMP (in resuspension buffer, final concentration 1.0 mM) in a 1 :2: 1 dilution (by volume). Twenty microliters (20 μl) of the solution containing 0.25 milligram per milliliter (mg/ml) protein were then plated into wells of a 48 well culture plate (Corning Inc., Corning, NY). The plate was centrifuged at 400 g for 2 minutes at room temperature to attach the cilia to the bottom of each well. A first measurement was taken (time t = 0) and 20 μl of ATP in resuspension buffer (Sigma, MO) (final concentration 1.25 . millimolar (mM)) were added to activate ciliary beating, typically at a frequency of 8-10 Hz. Test solutions containing antibodies or peptides in resuspension buffer were added at time 3 minutes, after three readings of activated cilia. Measurements of CBF were made at intervals of 1 minute for 15-20 minutes.

ATP depletion occurred at approximately 25 minutes after ATP addition. Reactivation of cilia was possible with further ATP addition, but was performed for control reasons only. For CBF measurements, antibodies and peptides were diluted in resuspension buffer. CBF was measured using the rapid automated digital analysis system described earlier (Sisson, JH et al., 2003, J. Microsc, 21 1 : 103-11 1). Cilia were visualized using phase contrast microscopy (Olympus IMT-2 inverted phase contrast microscope, Olympus America Inc., Melville, NY) directly connected to a digital camera (Kodak Megaplus ES 310 analog/digital video camera; Eastman Kodak Motion Analysis System Division, San Diego, CA) and a PC workstation (Dell Inc., Round Rock, TX). CBF was determined using Fourier analysis of the entire field of view from the digitized video.

Generation of antibodies against β tubulin isotypes Antibodies against the C-termini of β tubulin isotypes were raised in mouse hybridoma cells and purified as previously described (Banerjee, A et al., 1990, J. Biol. Chem., 265: 1794-1799; Banerjee, A et al., 1992, J. Biol. Chem., 267:5625-5630; Banerjee, A et al., 1988, J. Biol. Chem., 263:3029-3034; Roach, MC et al., 1998, Cell Modi. Cytoskeleton, 39:273-285). Each monoclonal antibody was prepared to an epitope unique to the C-terminus of that isotype. The C-termini of βlVa and βlVb are identical in amino acid sequence and therefore the antibody against βlV tubulin was unable to discriminate between them. All antibodies used in this study were purified monoclonal IgG class 1 antibodies except for anti βll and βlll tubulin, which were IgG2b (Table 2). The antibody against the βV tubulin C-terminus has not been previously reported (A. Banerjee, Deparment of Biochemistry, University of Texas Health Science Center at San Antonio). The monoclonal antibody SHM 12Gl 1 specific for mouse βV tubulin was generated by immunizing mice with the C-terminal peptide EEEINE (SEQ ID NO: 16). The peptide, coupled to keyhole limpet hematocyanin (KLH), was used to immunize mice. The antibody was purified from the hybridoma supernatant using a protein-G- sepharose column. Initial immunization was performed with peptide-KLH while the subsequent immunizations were performed with peptide coupled to BSA, according to Banerjee (Banerjee, A et al., 1988, J. Biol. Chem., 263:3029-3034).

Other antibodies were commercially obtained. The details are summarized in Table 3 Antibodies were used at a concentration of 20 μg/ml, which was within the range of concentrations used in several previous studies that employed antibodies to interfere with ciliary and flagellar beating (Audebert, S et al., 1999, Eur. J. Biochem., 261:48-56; Bre, MH et al., 1996, J. Cell Sci., 109 (Pt 4):727- 738; Cosson, J et al., 1996, Cell Motil. Cytoskeleton, 35: 100-1 12; Gagnon, C et al., 1996, J. Cell Sci., 109 (Pt 6):1545-1553). Controls to assure specific antibody binding to axonemal tubulin were performed by immunohistochemistry and immunoblotting.

Table 3. Epitopes of Antibodies

Ul

Visualization of antibody-labeled bovine cilia

Isolated cilia were plated onto concanavalin A-coated glass microscope slides (Sigma), allowed to settle for 1 hour and fixed using a solution of 1% paraformaldehyde in PBS. No permeabilizing step was necessary since the cilia are already demembraneted. Bound antibodies were visualized using goat anti- mouse secondary antibodies conjugated to Alexa 488 (Molecular Probes, Eugene, OR). Specimens were viewed using an Axioskop II microscope (Carl Zeiss, Jena, Germany) equipped with 4Ox and 10Ox objectives and captured using a Spot RT digital camera (Diagnostic Instruments, Sterling Heights, MI). Images were prepared for presentation using Photoshop (Adobe Systems, San Jose, CA). Negative controls were performed by omitting the primary antibody. Positive controls for the isotype-specific β tubulin antibodies were performed by staining organ of Corti tissue. The results seen were in concordance with previous observations (Hallworth, R et al., 2000, Hear. Res., 148: 161-172; Hallworth, R et al., 2000, Hear. Res., 139:31-41 ; Vent, J et al., 2005, J. Cell Sci., 1 18:4333-4341).

Immunoblotting

Immunoblots were performed under standard conditions with all antibodies used in the above experiments. A uniform amount of homogenized axonemal proteins (15 μg) was loaded onto each lane of a 10% polyacrylamide gel (Biorad, Hercules, CA). The protein content had been determined in a Bradford dye assay prior to aliquoting the extracted cilia using a microplate reader and protein (BSA) standards (MPM, microplate reader, Biorad). The lanes were run at 100 Volts (V) and 250 milliamps (mA) for 1 hour in sodium dodecyl sulfate electrophoresis running buffer. Proteins were then transferred from the gels to nitrocellulose sheets at 100 V and 100 mA for 1 hour in a transfer buffer containing Trizma base and glycine (Sigma). The nitrocellulose sheets containing the transferred protein lanes were then cut into strips and were exposed to one of primary antibodies overnight at 4°C. After thorough rinsing with 1% milk in PBS, the secondary antibody (anti- mouse IgG linked to biotin, Cell Signaling Technology, Beverly, MA), was added and the strips were incubated on a rocker for 1 hour at room temperature. The protein strips were then rinsed three times in 1% milk in PBS for 10 minutes

and twice in PBS for 10 minutes. They were then treated with SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Inc., Rockford, IL) for 3 minutes and exposed to a CL-XPosure blue Xray film (Pierce Biotechnology, Inc., Rockford, IL) for three seconds.

Generation of peptides

Axonemal motif peptides were synthesized using a previously published solid phase method (Taylor, CK et al., 2005, J. Pept. Res., 65:84-89). Briefly, N-α- butyloxycarbonyl (Boc)-amino acid derivatives (Bachem Biosciences Inc., King of Prussia, PA and Chemlmpex, Wood Dale, IL) were coupled to methylbenzhydryl amine resin (Bachem Biosciences Inc., King of Prussia,PA) using the coupling reagent 2-(lH-benzotriazole- l-yl)-l,l,3,3- tetramethyluronium hexafluorophosphate in excess base. All coupling yields were >99% as determined by the quantitative ninhydrin method (Sarin, VK et al., 1981 , Anal. Biochem., 117: 147-157). Boc groups were removed in trifluoroacetic acid (TFA, Acros Organics, Pittsburgh, PA). Peptides were cleaved from the resin using a mixture of trifluoromethanesulphonic acid/TFA/ethanedithiol/thioanisole (1/9/0.5/1 , v/v) and immediately desalted on a BioGel P6 column (90 x 2.5 cm) (Bio Gel P6, Bio Rad, Hercules, CA) in 5% aqueous acetic acid. The desalted material was loaded on to a Vydac Cl 8 HPLC column (25x2.5 cm) (Vydac Cl 8 column, The Sep/a/ra/tions Group, Hesperia, CA) previously equilibrated with a mixture of water and acetonitrile (9/1, v/v) (Fisher Scientific, Pittsburgh, PA) containing 0.1 % TFA. The concentration of acetonitrile in the eluent was increased from 10 to 40% over 50 minutes to elute the peptides from the column. Fractions containing the desired peptides were pooled and lyophilized. Peptides were judged to be >95% pure by analytical RP-HPLC and possessed satisfactory amino acid compositions. The peptides containing the sequences EGEAEEE (SEQ ID NO: 18), EGEGEEE (SEQ ID NO: 19), EDEDEGE (SEQ ID NO:20) and GEMYEDD (SEQ ID NO:1 1) were custom made peptides purchased from Bachem

Biosciences Inc. (King of Prussia, PA). The C-terminal tail peptides were the peptides against which the β tubulin isotype specific antibodies were raised (see Table 1 and Table 2 for sequences). Their synthesis is described elsewhere (Banerjee, A et al., 1990, J. Biol. Chem., 265:1794-1799; Banerjee, A

et ah, 1992, J. Biol. Chem., 267:5625-5630; Banerjee, A et al., 1988, J. Biol. Chem., 263:3029-3034; Renthal, R et al., 1993, Cell Motil. Cytoskeleton, 25: 19- 29). Unless otherwise indicated, chemicals were purchased from Sigma-Aldrich Co. (St Louis, MO).

Effect of antibodies against βl, βlV and βV tubulin block ciliary beating

It was hypothesized that the C-terminus of β tubulin has functional importance in ciliary beating. Thus, blocking this domain with antibodies should interfere with ciliary beating in isolated bovine cilia. On application of ATP solution (final concentration 1.25 mM) after the initial reading at time t = 0, isolated cilia began to beat continuously at about 10 Hertz (Hz). The CBF reached a typical steady state value by time t = 3 minutes, at which time solutions containing the antibody were added at a final concentration of 20 μg/ml (equivalent to 0.33 μM). Axonemes maintained a steady CBF until ATP was depleted, typically after 20 minutes. Fig. 1 A shows the effect of application of antibodies on CBF. Each data point is plotted as the mean and one standard error of the mean for at least six experiments. Antibodies against βl, βlV and βV tubulin reduced CBF to zero within 8-10 minutes. By contrast, antibodies against βll and βlll tubulin, as well as the buffer only controls (negative control), did not affect CBF. These results indicate that the isotype-specific antibodies against βl, βlV and βV tubulin interfere with the motility-determining structures of microtubules, and that only those isotype-specific antibodies raised against the C-terminal sequences of βl, βlV and βV tubulin are able to block ciliary beating. The effect of the antibodies on CBF is concentration dependent. It was thus hypothesized that the inhibition of ciliary beating by antibodies would be specific and thus concentration dependent. To test this hypothesis, the effect of antibodies at different concentrations was examined. When the concentration of antibodies against βl, βlV and βV tubulin was decreased in a logarithmic manner, the time taken for reduction of CBF to zero was increased. Fig. IB shows the effect of antibody against βlV tubulin on CBF at varying the concentrations (20 μg/ml, 2 μg/ml and 0.2 μg/ml final concentrations, equivalent to 0.33 μM, 0.033 μM and 3.3 nM, respectively). Comparable results were obtained with antibodies against βl and βV tubulin (data not shown). Reduction of antibody concentration by at least two orders of

magnitude from the initial concentration was required to reduce the effect of the antibodies to control levels.

Effect of preincubation of axonemes with antibodies inhibited reactivation As a further test of the specificity of the antibody effect, cilia were pre- incubated with a molar excess (10 μM) of antibody for 1 hour on ice. The high concentration was chosen to assure all epitopes would be bound by antibody. After equilibration to room temperature, ATP was added to activate ciliary beating. Normal activation to a CBF of 10 Hz was achieved in all samples except the ones containing antibodies against βl, βlV and βV tubulin, which did not activate.

Effect of antibodies against other epitopes of β tubulin, or against a tubulin on CBF It was hypothesized that the effects of the antibodies against the C- termini of βl, βlV and βV tubulin on CBF were specific to the C-terminus of tubulin isotypes. To test this hypothesis, monoclonal antibodies against other epitopes of α and β tubulin were applied. Fig. 1C shows the effect on CBF of an antibody against a C-terminal, non-isotype-specific epitope of α tubulin (DMlA) and an antibody against a non-isotype-specific epitope (within position 281-446) of β tubulin (TUB 2.1). The antibodies had no effect on CBF at the same concentration as the maximum concentration used in the above described studies. Even higher concentrations (200 μg/ml or 0.66 μM) did not affect CBF (data not shown). Fig. ID shows the effect of two monoclonal non- isotype specific antibodies raised against the C- and N-termini of β tubulin, respectively. They had no effect on CBF at comparable concentrations. These experiments further support the inference that the effects of the antibodies against βl, βlV and βV tubulin were specific to the binding sites of those antibodies, and that axonemal motility is dependent on the C-terminal epitope of β tubulin isotypes or its direct proximity against which the antibodies are directed. Furthermore, the results indicate that there is no steric hindrance by ineffective antibodies, even against the C-terminus of β tubulin, to the dynein binding site. They also exclude any non-specific inhibitory effect of the antibodies.

Specific peptides reduce CBF

Nielsen et al. (Nielsen, MG ct ah, 2001 , Curr. Biol., 11 :529-533) hypothesized that a certain amino acid sequence, the axonemal motif, is required for ciliary function and assembly. The axonemal motif hypothesis was thus tested using synthesized heptapeptides containing the axonemal motif or amino acid sequences in corresponding positions of mammalian βl, βll, βlll, βlV and βV tubulin, hypothesizing that the peptides would mimic the C-terminus of β tubulin and thus competitively inhibit beating. Their effects were compared to the effects of the C-terminal tail peptides against which the antibodies were raised. Fig. 2A-E and Table 3 show the results of these experiments. Each plot shows the effect on CBF of the axonemal motif peptide for each isotype (open triangles), compared to the effect of the corresponding C-terminal tail peptide (open circles), at the same molar concentrations of peptide (final concentration 0.33 μM) as was used for the antibodies. Each plot also shows the average of a series of negative control experiments (filled squares). The amino acid sequences of the C-terminal peptides are given in Table 1. These results of these experiments are shown in Fig. 2(A-E) and are summarized in Table 4 In general, the axonemal motif peptides were effective in reducing CBF while the C-terminal tail peptides were much less so. For example, the βlV and βV tubulin axonemal motif peptides completely abolished ciliary beating in 15 minutes or less, while the corresponding C-terminal tail peptides had only weak effects on CBF (Fig. 2D,E). Both the axonemal motif and C-terminal tail peptide of βlll tubulin were ineffective in reducing CBF (Fig. 2C), as expected since βlll tubulin is not present in the bovine cilia preparation. However, the axonemal motif peptide of βll tubulin, which is also not present in bovine tracheal cilia, was effective in abolishing ciliary beating, whereas the corresponding C-terminal tail peptide had only marginal effect (Fig. 2B). Further, both βl tubulin peptides were effective in reducing CBF (Fig. 2A). It was observed that the content of acidic amino acids (E and D) accounts for much of the inhibitory effect on CBF. The axonemal motif-like peptides generally had five acidic amino acids (with the exception of the peptides of βll tubulin and βlll tubulin that have four), while the C-terminal tail peptides have four acidic acids (with the exception of the peptides of βl tubulin and βlll tubulin that have

five and one, respectively). The C-terminal tail peptide of βl tubulin (EEAEEEA (SEQ ID NO:8)) was noted to be equally effective in reducing CBF as its axonemal motif peptide (EEDFGEE (SEQ ID NO:7)).

Table 4 Effects of Peptides on CBF

-, no effect; +, some inhibitory effect; ++, moderate inhibitory effect; +++, strong inhibitory effect leading to reduction of CBF to 0 Hz.

The effect of the axonemal motif peptide is concentration dependent It was hypothesized that the effect of the βlV tubulin axonemal motif peptide EGEFEEE (SEQ ID NO: 13) is specific and therefore concentration dependent. To test this, the peptide was added to the activated cilia preparation at concentrations of 0.33 μM, 0.033 μM and 3.3 nM. The highest concentration of peptide reduced CBF the fastest, while the lowest concentration was essentially ineffective (Fig. 2F).

The central phenylalanine is important in the axonemal motif sequence

It was observed that the axonemal motif peptides that reduced CBF contained, in addition to several acidic residues, a central F at position 436. Also, F436 is not present in the axonemal motif peptide of βlll tubulin, which did not reduce CBF. To test the hypothesis that F436 is important, F436 in the axonemal motif sequence (EGEFEEE) (SEQ ID NO: 13) was replaced with alanine, resulting in the sequence EGEAEEE (SEQ ID NO: 18). As shown in Fig. 3A, this peptide had no effect on CBF, demonstrating the importance of F436.

Axonemal motif -like sequences in the C-terminus of a tubulin are not involved in ciliary beating

An E-rich sequence similar to the axonemal motif is present in the C- termini of several β tubulin isotypes between positions 441 and 447, but these sequences lack the central F (Table 4). To test the hypothesis that the C-terminus of α tubulin is not involved in ciliary beating, two peptides were synthesized that represented the sequences of four α tubulin isotypes: EGEGEEE (SEQ ID NO: 19) (α II, α III) and EDEDEGE (SEQ ID NO:20) (αl, αlV) (Luduena, RF et al., 1975, Ann. N. Y. Acad. Sci., 253:272-283; Luduena RF et al., 2005, The isotypes of tubulin: distribution and functional significance. In Microtubules (ed. T. Fojo). Totowa, NJ: Humana Press (in press); Luduena RF, et al., 2005, The post-translational modifications of tubulin. In Microtubules (ed. T. Fojo). Totowa, NJ: Humana Press (in press); Luduena RF, et al., 2005, The tubulin superfamily. In Microtubules (ed. T. Fojo). Totowa, NJ: Humana Press (in press)). Neither peptide affected ciliary beating at comparable concentrations to previous experiments, supporting the antibody observations that the C-terminus of α tubulin may not be directly involved in ciliary beating (Fig. 1C,D and Fig. 3B).

Antibodies are specific for axonemal tubulin

It was hypothesized that the effects of antibodies against βl, βlV and βV tubulin on ciliary beating were specific for binding at the β tubulin isotype specific epitopes and not due to unspecific inhibition of beating. To test this hypothesis, all antibodies were applied to fixed preparations of bovine cilia and

processed for immunofluorescence as described herein. Antibodies against βl, βlV and βV tubulin, which blocked ciliary beating, labeled cilia (Fig. 4). The same was observed for the antibodies against the C- and N-termini of α tubulin, as well as for antibodies against conserved, non-isotype-specific epitopes in the α and β tubulin protein (DMlA and TUB 2.1). However, they did not inhibit ciliary beating, making steric hindrance as a reason for CBF inhibition by anti βl, βlV and βV tubulin antibodies unlikely. Antibodies against β tubulin isotypes not present in tracheal cilia (anti βll and anti βlll tubulin) did not label isolated cilia in the described preparation (Fig. 4A,B). Immunoblots of purified de-membranated cilia with all of the above mentioned primary antibodies indicated that labeling is restricted to a single band of molecular mass close to 55 kDa (Fig. 4B), the expected molecular mass of β tubulin (Luduena, RF, 1998, Int. Rev. Cytol., 178:207-275). The amino acid sequence difference between β tubulin isotypes is so small that the isotypes are indistinguishable by molecular mass alone. Strong labeling appeared with anti βl, DMlA and Tub 2.1

(antibodies against conserved, non-isotype specific epitopes of α and β tubulin) was as well as the C-and N-terminus specific antibodies against β tubulin. Antibodies against βll and βlll tubulin did not label the axonemal proteins at all, because βll and βlll tubulins are not synthesized in bovine tracheal cilia. A weak band occurred at 55 kDa with labeling by antibodies against βlV and βV tubulin. Controls for the well-established antibodies against βϋ and βlll tubulin were performed by staining the organ of Corti.

Data according to Luduena RF et al., 2005, The isotypes of tubulin: distribution and functional significance. In Microtubules (ed. T. Fojo). Totowa, NJ: Humana Press (in press).

DISCUSSION

The axonemal motif is important for ciliary function

The findings suggest that the axonemal motif, EGEFEEE (SEQ ID NO: 17), is important for ciliary function and dynein binding. Generally, the results were consistent with the number of acidic amino acids (five E or Ds) determining its effectiveness, with the exception of the axonemal motif-like peptide of βll tubulin, which contains only four acidic amino acids but is equally effective as the axonemal motif. Mizuno et al. (Mizuno, N et al., 2004, EMBO J., 23:2459-2467) inferred that dynein and kinesin share an overlapping binding site on the tubulin C-terminus, indicating that dynein binds to either α or β tubulin, but not both. These results were suggestive of β tubulin being the target protein. Skiniotis et al. (Skiniotis, G et al., 2004, EMBO J., 23:989-999) further support (Gee, MA et ah, 1997, Nature, 390:636-639)the importance of the tubulin C-terminus by its subtilisin digestion. However, this does not exclude the possibility that other regions of tubulin are also required, analogous to the weak and strong binding states of kinesin (Skiniotis, G et ah, 2004, EMBO J., 23:989-999). Nevertheless, findings from kinesin cannot definitively be applied to dynein. Kinesin contains a lysine (K)-rich sequence on its tubulin- binding site, termed the K-loop, which acts as a counterpart to the acidic C- terminus of tubulin (E-hook) (Okada, Y et ah, 2000, Proc. Natl. Acad. Sci. U. S. A, 97:640-645). No such sequence exists in dynein (Asai, DJ et ah, 2001 , Trends Cell Biol., 1 1 :196-202; Gee, MA et ah, 1997, Nature, 390:636-639). Inferences drawn from the interaction of kinesin with tubulin to dynein must be made carefully.

The axonemal motif sequence pivots around the central F

It was found that, in general, C-terminal acidic residues (E or D) are required for ciliary beating. The higher the content of acidic amino acids in the peptide, the stronger was the inhibitory effect on beating. Further, it was shown that this region must pivot around the central F436 found in some β tubulin

isotypes. However, the study referred to earlier by Okada and Hirokawa did not evaluate dynein and did not focus on the isotype-specific differences in the C- termini of β tubulins (Okada, Y et ah, 2000, Proc. Natl. Acad. Sci. U. S. A, 97:640-645). In this study, the further requirement of a central F was established in addition to acidity as a key characteristic for axonemal function. In general, the C-terminal tail peptides, which did not contain F, were less effective than the axonemal motif peptides, with the exception of the peptides for βl and βV tubulin, which were equally effective. This hypothesis is supported by the lack of effect of the two β tubulin peptides (Fig. 3) which despite being highly acidic lack the central F.

a tubulin is not directly involved in ciliary beating

The evidence that α tubulin is not directly involved in ciliary beating is, on first inspection, surprising. Three different antibodies against α tubulin, two of which were directed against a conserved, non-isotype specific C-terminal amino acid sequence (419-435 and 426-450), had no effect on ciliary function. Furthermore, peptides of the α tubulin axonemal motif sequence did not affect CBF. These results are consistent with recent literature that suggests no involvement of α tubulin in ciliary beating (Audebert, S et al., 1999, Eur. J. Biochem., 261 :48-56; Cosson, J et al., 1996, Cell Motil. Cytoskeleton, 35: 100- 1 12). Some earlier investigations favored α tubulin being important for ciliary function (Gagnon, C et al., 1996, J. Cell Sci., 109 ( Pt 6): 1545-1553; Goldsmith, M et al., 1995, Biochem. Cell Biol., 73:665-671 ; Goldsmith, M et al., 1991 , Cell Motil. Cytoskeleton, 20:249-262; Hirose, K et al., 1999, Cell Struct. Funct., 24:277-284). Goldsmith et al. suggest the involvement of both α and β tubulin for dynein binding (Goldsmith, M et al., 1995, Biochem. Cell Biol., 73:665-671 ; Goldsmith, M et al., 1991 , Cell Motil. Cytoskeleton, 20:249-262). The role of α tubulin might lie in other, yet undetermined functions. For example, α tubulin may function in binding of microtubule-associated proteins (MAPs) (Rodionov, VI et al., 1990, J. Biol. Chem., 265:5702-5707) or interact with dynein for control of microtubule dynamics (Hunter, AW et al., 2000, J. Cell Sci., 1 13 Pt 24:4379-4389). However, those studies were conducted on cytoplasmic dynein. Hunter and Wordeman showed that the C-terminus of tubulin is necessary for MAP binding and further suggested a tubulin-binding site of dynein outside the

C-terminus. Hoenger et al. describe that each kinesin dimer occupies two microtubule binding sites (Hoenger, A et al., 2000, J. MoI. Biol., 297: 1087- 1 103). That might be true for dynein also, but has not been investigated so far. The periodicity of the αβ tubulin heterodimer in the microtubule is 8 nm. In cytoplasmic dynein, the step size has been proposed to be dependent on cargo load and to have a minimum of 8 nm (Mallik, R et al., 2004, Nature, 427:649- 652). If the step size of axonemal dynein is similar, dynein may skip α tubulin and interact only with the C-terminus of β tubulin. The sequences of the peptides EDEDEGE (SEQ ID NO:20) (in αl and αlV tubulin) and EGEGEEE (SEQ ID NO: 19) (in all and αlll tubulin) were derived from Luduena and Banerjee (Luduena RF et al., 2005, The isotypes of tubulin: distribution and functional significance. In Microtubules (ed. T. Fojo). Totowa, NJ: Humana Press (in press)). Due to the absence of isotype specific antibodies for α tubulins, previous information on the possible distribution of α tubulin isotypes was obtained by in situ hybridization. The presence of these α isotypes (I-IV) in the bovine tracheal cilia preparation is plausible but not established (Luduena RF et al., 2005, The isotypes of tubulin: distribution and functional significance. In Microtubules (ed. T. Fojo). Totowa, NJ: Humana Press (in press)), thus there is a small possibility that the peptides have no effect due to the absence of these α tubulin isotypes in bovine cilia. However, antibodies against various non- isotype-specific epitopes of α tubulin, including the C-terminus, also did not affect CBF.

Posttranslational modifications may alter the secondary structure of the C- terminus

Posttranslational modifications (PTM) in tubulins occur mainly at the highly flexible C-terminus (Luduena, RF, 1998, Int. Rev. Cytol., 178:207-275). The most recently discovered PTM, polyglycylation, has been shown to occur in axonemal tubulin from Paramecium to sea urchin and mammalian spermatozoa (Bre, MH et al., 1996, J. Cell Sci., 109 ( Pt 4):727-738). Bre et al also suggested an involvement of polyglycylated tubulin in axoneme motility since AXO 49 and TAP 952, monoclonal antibodies against mono- and polyglyclyated C- terminal peptides from Paramecium axonemal tubulin, specifically inhibited the reactivated motility of sea urchin spermatozoa. Polyglycylation occurs at the C-

terminus and is highly variable in its amount (up to 32 glycines on a side chain off the γ- carboxyl chain of glutamic acids E435, E437 and E438). Polyglycylation has not yet been shown to be functionally required, but may affect the secondary structure of the highly flexible C-terminus in tubulin. Gagnon et al. reported that a different PTM, polyglutamylation, in the lateral chain of α tubulin plays a dynamic role in spermatozoan motility (Gagnon, C et al., 1996, J. Cell ScL, 109 ( Pt 6): 1545-1553). Possibly due to steric hindrance by the large antibody, Gagnon et al. could not determine the amino acid sequence required for ciliary beating.

Isotype specificity of β tubulin in axonemes

This leads back to the initial question of the existence of functional correlations of β tubulin isotypes: why do some cells synthesize β tubulin isotypes in one pattern and others synthesize different isotypes? Why, if the isotypes are so similar in amino acid sequence, is there still a requirement for the different isotypes? The answer may lie in the highly variable C-terminus. It was previously found that βl and βlV tubulin are synthesized by all ciliated cells types tested in the gerbil (Jensen-Smith, HC et al., 2003, Cell Motil. Cytoskeleton, 55:213-220). It was inferred that both isotypes are required for axonemal assembly and/ or function. However, those observations were based on immunohistochemistry alone. βV tubulin was recently found to be in some but not all motile cilia. Therefore, βV tubulin may be capable of supporting ciliary beating, but may not be absolutely required for ciliary function, βll tubulin has so far not been detected in cilia. However, its axonemal motif-like sequence differs only in the first amino acid from the axonemal motif sequence of βlV tubulin (E433 to Q433). Further, the axonemal motif peptide of βll tubulin was nearly as potent as that of βlV tubulin in blocking ciliary beating. Is βll tubulin incompatible with the correct axonemal assembly or function? A change in only position 433 from glutamate to glutamine is observed, resulting in a removal of one charge in βll tubulin. This may indicate that the secondary structure of the synthetic peptide resembles the axonemal motif peptide more closely that does the corresponding sequence in βll tubulin. It is worth noting that the unusual β tubulin isotypes βll and βlll are in some circumstances capable of supporting axonal assembly, if not motility. In

the globose basal cells of the nose (the olfactory stem cells) βl, βll and βlll tubulin are synthesized and incorporated into microtubules. As soon as the cell matures and develops long, immotile sensory cilia, βlV tubulin is also synthesized (Woo, K et al., 2002, Cell Tissue Res., 309:331-335). The isotypes do not compartmentalize, that is, all synthesized isotypes are incorporated into all microtubule structures in the cell, including the cilia. Thus βll tubulin and βlll tubulin are at least capable of assembling into immotile cilia. It remains to been seen if βll tubulin and βlll tubulin are capable of supporting ciliary motility and the assembly into a 9+2 structure, or if they disrupt these.

The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

SEQUENCE LISTING FREE TEXT

SEQ ID NO: 1 EGEFXXX, peptide where X is either glutamic (E) or aspartic (D) acid)

SEQ ID NO:2 EEEDFGEEAEEEA peptide SEQ ID NO:3 EQGEFEEEEGEDEA peptide

SEQ ID NO:4 EGEMYEDDEEESES peptide

SEQ ID NO:5 EEGEGEEEAEEEVA peptide

SEQ ID NO:6 GEEAGEDEDEEEIN peptide

SEQ ID NO:7 EEDFGEE peptide

SEQ ID NO:8 EEAEEEA peptide SEQ ID NO:9 QGEFEEE peptide SEQ ID NO: 10 EGEDEA peptide SEQ ID NO: 1 1 GEMYEDD peptide SEQ ID NO: 12 SESQGPK peptide SEQ ID NO: 13 EGEFEEE peptide SEQ ID NO: 14 EAEEEVA peptide SEQ ID NO: 15 EEAFEDE peptide SEQ ID NO: 16 EEEINE peptide SEQ ID NO: 17 EGEFEEE peptide SEQ ID NO: 18 EGEAEEE peptide SEQ ID NO: 19 EGEGEEE peptide SEQ ID NO:20 EDEDEGE peptide SEQ ID NO:21 SEAREDMAALEKDYEEV peptide SEQ ID NO:22 DSYEDEDEGEE peptide SEQ ID NO:23 DSVEGEGEEEGEEY peptide SEQ ID NO:24 DSVEGEGEEEGEEY peptide SEQ ID NO:25 DSYEDEDEGEE peptide SEQ ID NO:26 DSVEAEAEEGEEY peptide SEQ ID NO:27 DSFEEENEGEEF peptide SEQ ID NO:28 SESQGPK peptide SEQ ID NO:29 LVSEYQQYQDATAEEEGEMYEDDEEESEAQGPK peptide

SEQ ID NO:30 LVSEYQQYQDATAEEEEDFGEEAEEEA peptide

SEQ ID NO: 31 LVSEYQQYQDATAEEEEDFGEEAEEEA peptide SEQ ID NO:32 LVSEYQQYQDATAEEEGEFEEEAEEEVA peptide SEQ ID NO:33 LVSEYQQYQDATAEQGEFEEEAEEEVA peptide