CBS patent_US20110105382

US 20110105382A1

(19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0105382 A1

Husain et al. (43) Pub. Date: May 5, 2011

(54) CALMODULIN-BINDING PEPTIDES THAT C12N 5/09 (2010.01) REDUCE CELL PROLIFERATION IN C12N 5/071 (2010.01) CANCER AND SMOOTH MUSCLE A61P 35/00 (2006.01) PROLIFERATION DISEASES A61P 9/10 (2006.01)

A61P 9/12 (2006.01) (76) Inventors: Mansoor Husain, Toronto (CA); C0711 21/00 (200601)

Jaehyun Choi, Toronto (CA) C07K 16/18 (200601) A61P 21/00 (2006.01)

(21) APP1-NO-I 12/674,368 A61P 17/00 (2006.01) _ A61P 1/00 (2006.01)

(22) PCT Flledl Aug. 19, 2008 A611) 13/10 (200601)

(86) PCT No.: PCT/CA2008/001474 § 371 (c)(1), (52) US. Cl. ........ .. 514/1.9; 530/326; 530/327; 530/328;

(2), (4) Date: Jun. 2, 2010 530/329; 530/330; 530/324; 514/21.4; 435/375; 514/19.3; 514/15.7; 536/235; 530/387.9;

Related US. Application Data 514/18_6

(60) Provisional application No. 60/957,361, ?led on Aug. 22’ 2007_ (57) ABSTRACT

_ _ _ _ The invention relates to an isolated peptide comprising all or Pubhcatlon Classl?catlon part of the amino acid sequence: GGAEFSARSR KRKAN

(51) Int, Cl, VTVFL QD (SEQ ID NO: 2), Wherein the peptide reduces A61K 38/17 (200601) cell proliferation. The invention also includes variants, such C07K 14/47 (200601) as SEQ ID NO:3-5 and fragments of at least 5 amino acids of C07K 7/08 (200601) SEQ ID NO:2-5 that reduce cell proliferation. The peptides C07K 7/06 (200601) are useful for treating cancer and diseases characterized by C07K 19/00 (200601) smooth muscle proliferation, such as restenosis.

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GGSDLSVRSRKRKANVAVFLQD SEQ ID NO: 3 (mouse)

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US 2011/0105382 A1

CALMODULIN-BINDING PEPTIDES THAT REDUCE CELL PROLIFERATION IN CANCER AND SMOOTH MUSCLE PROLIFERATION DISEASES

FIELD OF THE INVENTION

[0001] The invention relates to peptides for use in treatment of cancer and vascular disease.

BACKGROUND OF THE INVENTION

[0002] There are a number of diseases caused by cell pro liferation Which are not currently treated properly and safely With conventional medicine. For example, cancer is caused by cells that proliferate uncontrollably. Radiation treatment and surgery are often used for cancer treatment even though these treatments produce severe side effects. Despite recent advances in cancer chemotherapy, radiation treatment and surgery remain the mainstays of cancer treatment. Cell cycle inhibitors are an example of a type of chemotherapy drug that have been used to treat cancer, but these compounds have been shoWn to cause severe toxicity With poor selectivity. [0003] Vascular smooth muscle cell proliferation is respon sible for a number of diseases, such as restenosis folloWing balloon angioplasty. Restenosis, is a relatively frequent (-10%) consequence of balloon angioplasty procedures per formed on occluded or narroWed coronary arteries and/or coronary artery bypass grafts (“CABG”). Angioplasty has over-taken CABG surgery as the most common heart proce dure performed in the World today but current therapies have had limited success in preventing restenosis. As Well, con cerns surrounding the safety of radioactive stents, and stents that elute chemotoxic agents, highlight the need for alterna tive strategies aimed at treating this disease. [0004] Advances have been made in understanding the bio chemical interactions that regulate cell division. Calmodulin (“CaM”), a small acidic protein of 16.7 kDa (Genbank acces sion no. for human CaM is CAA36839), is a principal Ca2+ sensor in eukaryotic cells that contains four EF-hand Ca2+ binding motifs. Upon binding With Ca2+, Ca2+/CaM per forms a large conformational change of its highly ?exible ot-helical linker resulting in its binding to a variety of target proteins. These are collectively termed CaM-binding pro teins, through Which Ca2+-sensitivity is expressed in a vari ety of cell biological functions including ion channel func tion, gene regulation, smooth muscle contraction, and cell motility. A role of CaM as a regulator of cell cycle progression has also been recognized. A direct interaction betWeen the major calcium {Ca2+} signal-transducing protein calmodu lin (CaM), and the critical regulator of cell cycle progression cyclin E, is necessary for Ca2+-sensitive cyclin E/CDK2 activity during G1 to S phase cell cycle progression in vas cular smooth muscle cells (“VSMC”). The human cyclin E gene Was cloned, sequenced and functionally characterized as a cell cycle regulatory protein betWeen 1991 and 1992 (LeW Cell 91; Koff Cell 91; Koff Science 92; Dulic Science 92; Matsushime Cell 1991); Genbank accession no. for human cyclin E is NP 476530). This metabolic interaction is related to proliferation of vascular smooth muscle cells and cancer cells, hoWever, there are no medicines Which target this inter action selectively in order to treat proliferative disorders.

SUMMARY OF THE INVENTION

[0005] The invention relates to isolated peptides for use in treatment of cancer and vascular disease by disrupting calm

May 5,2011

odulin binding to cyclin E protein. The inventors have syn thesiZed useful isolated peptides With the sequence shoWn in FIG. 9 (SEQ ID NOS:2-5) as Well as shorter peptides based on this sequence. The inventors provide isolated peptides as small as 5 amino acids that provide cell proliferation inhibi tion activity (“CBS activity”). [0006] The peptides of the invention reduce proliferation of primary vascular smooth muscle cells and cancer cells by causing cell cycle arrest. They do not cause cytotoxicity (i.e. cell death) and do not interfere With other CaM-signaling pathways-they act selectively through the CaM-cyclin E interaction. These characteristics set the peptides apart from other existing cell cycle inhibitors and make them useful for therapeutic treatments that inhibit cell proliferation. CBS peptides have several speci?c advantages, including (a) high selectivity; Working in a cyclin E-speci?c/ dependent manner, and (b) loW toxicity; not causing cell death and not inhibiting basal CDK2 activity. [0007] The invention relates to isolated peptides capable of reducing cell proliferation and comprising all or part of (SEQ ID NOS:2-5). In one embodiment, the peptide reduces smooth muscle cell or cancer cell proliferation. The isolated peptides are optionally synthetic peptides or recombinant peptides. [0008] In another embodiment, the peptide has three con secutive hydrophobic residues located Within ?ve amino acids of the C-terminus. Optionally, at least one of the hydro phobic residues comprises a leucine residue. Optionally, the leucine residue is located Within 5 amino acids or Within 3 amino acids of the C-terminus.

[0009] In a variation of the invention, the peptide comprises a truncated amino acid sequence of the peptide of any one of (SEQ ID NOS:2-5) or a fragment of the amino acids of the peptide of (SEQ ID NO:2). Optionally, the truncated peptide contains amino acids 2-21, 3-20, 4-19, 5-18, 6-17, 7-16, 8-15, 9-14, 12-20, 14-20 or 16-20 ofthe peptide of (SEQ ID NOS: 2-5). The fragment optionally contains 5-10, 10-15, 15-20 or 20-22 amino acids of the peptide of (SEQ ID NOS:2-5). In another variation, the peptide comprises a sequence falling Within the formula V-X1-X2-F-L, Wherein X1 is optionally a hydrophobic amino acid or a neutral amino acid (eg. selected from the group ofV, I, L, F, W, C, A,Y, H, T, S, P, G, R or K), typically T, A, P and Wherein X2 is optionally a hydrophobic amino acid or a neutral amino acid (eg. selected from the group ofV, I, L, F, W, C, A, Y, H, T, S, P, G, R or K), typically V or T. The peptide optionally contains 5-10, 10-15, 15-20 or 20-22 or 23-50 amino acids.

[0010] In another variation of the invention, the peptide comprises at least 5, 6, 7, 8, 9 or 10 amino acids of the peptide of any one of SEQ ID NOS:2-5.

[0011] In another variation of the invention, the peptide contains at least 80% sequence identity With the amino acid sequence of SEQ ID NOS:2-5 and reduces cell proliferation. For example, the peptides having identity optionally contain all or part of the amino acid sequences of any of SEQ ID NOS:2-5.

[0012] In another embodiment of the invention, the peptide of the invention further comprises a TAT linker amino acid sequence, comprising all or part of RRRQRRKKRG, to increase the uptake of the peptide into a cell.

[0013] In another embodiment of the invention a pharrna ceutical composition comprises any of the peptides of the invention and a carrier.

US 2011/0105382 A1

[0014] The peptides may be modi?ed as described below to produce variants of the peptide that have desired activities. For example, the invention includes peptide sequences plus or minus amino acids at the amino and/or carboxy terminus of the peptide sequences. In another embodiment, the invention includes fusion proteins, comprising the CBS peptide or labeled CBS peptide. A peptide of the invention may include various structural forms of the primary CBS peptide Which retain biological activity. For example, a peptide of the inven tion may be in the form of acidic or basic salts or in neutral form. In addition, individual amino acid residues may be modi?ed by oxidation or reduction. [0015] The invention further relates to a method of reduc ing cell proliferation caused by cyclin E speci?c calcium/ calmodulin dependent CDK2 activity in the cell, comprising administering to the cell a peptide or pharmaceutical compo sition of the invention. Optionally, the cell is a cancer cell or a smooth muscle cell. The cancer cell is optionally a cervical cancer cell, an osteosarcoma cancer cell or a lung cancer cell.

[001 6] The invention further relates to the use of the peptide or the pharmaceutical composition of the invention for treat ment of cancer and to the method of treatment of cancer in a mammal Wherein the peptide or pharmaceutical composition is administered to the mammal. Optionally, the cancer com prises cancer cells undergoing calcium sensitive cyclin E protein mediated cell proliferation. The cancer is optionally cervical cancer, osteosarcoma or lung cancer.

[0017] The invention further relates to the use of the pep tides or the pharmaceutical composition of the invention for reducing proliferation of vascular smooth muscle cells and the method of reducing proliferation of vascular smooth muscle cells in a mammal in need thereof Wherein the peptide or pharmaceutical composition is administered to the mam mal. Optionally, the disorder comprises vascular smooth muscle cells undergoing calcium sensitive cyclin E protein mediated cell proliferation. Optionally, the peptide or phar maceutical composition inhibits CDK2 activity by inhibiting the binding of calmodulin to cyclin E protein. [0018] The invention further relates to the use of the pep tides or the pharmaceutical composition of the invention for the treatment of a vaso-occlusive disorder and to a method of treatment of a vaso-occlusive disorder in a mammal in need thereof Wherein the peptide or pharmaceutical composition is administered to the mammal. Optionally, the vaso-occlusive disorder comprises restenosis, Burger syndrome, atheroscle rosis, scleroderrna, Raynauds disease, hypertension pulmo nary hypertension or post-vascular surgery smooth muscle cell proliferation. The atherosclerosis optionally comprises coronary artery disease, peripheral artery disease or cere brovascular disease. The hypertension optionally is caused by smooth muscle cell proliferation after vascular surgery. The vascular surgery optionally consists of coronary angioplasty, coronary stent placement, coronary by-pass surgery, periph eral stent placement, vascular grafting, thrombectomy, vas cular angioplasty, and vascular stenting. Optionally, the dis order comprises vascular smooth muscle cells undergoing calcium sensitive cyclin E protein mediated cell proliferation. Optionally, the pharmaceutical composition is a stent. [0019] The invention further relates to the use of the pep tides or the pharmaceutical composition for the treatment of a visceral smooth muscle cell disorder and a method of treat ment of a visceral smooth muscle cell disorder in a mammal in need thereof Wherein the peptide or pharmaceutical com position is administered to the mammal. Optionally, the vis

May 5,2011

ceral smooth muscle cell disorder comprises in?ammatory boWel disease, boWel strictures, spastic bladder, urinary retention and uterine cramps. The smooth muscle cell prolif eration is optionally caused by vascular injury. Optionally, the disorder comprises vascular smooth muscle cells undergoing calcium sensitive cyclin E protein mediated cell proliferation. [0020] The invention further relates to an isolated nucleic acid comprising all or part of (SEQ ID NO:1) that encodes a peptide of the invention. The invention further relates to an isolated nucleic acid encoding all or part of any of SEQ ID NOS:2-5. The encoded peptide reduces smooth muscle cell or cancer cell proliferation. [0021] The invention further relates to an isolated antibody that selectively binds any one or more of the peptides of the invention. [0022] The invention further relates to a stent comprising any of the peptides or the pharmaceutical composition of the invention. [0023] Other features and advantages of the present inven tion Will become apparent from the folloWing detailed description. It should be understood, hoWever, that the detailed description and the speci?c examples While indicat ing preferred embodiments of the invention are given by Way of illustration only, since various changes and modi?cations Within the spirit and scope of the invention Will become apparent to those skilled in the art from the detailed descrip tion.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Embodiments of the invention Will be described in relation to the draWings in Which: [0025] FIG. 1 shoWs that the CaM-cyclin E interaction is reduced by the synthetic peptide of CaM-binding motif. [0026] FIG. 1A provides the sequences of the synthesiZed peptides. Human CBS 22 mer (SEQ ID NO:2): Isolated pep tide corresponds to sequence of the CaM binding region in human cyclin El. 5A: Same as human CBS 22 mer except for 5 alanine mutations at every hydrophobic residue. NC: nega tive control Which has the same length, 22 amino acids, as CBS (SEQ ID NO:2). [0027] FIG. 1B shoWs a histone H1 in vitro kinase assay from Gl/S-synchroniZed mouse VSMC in the presence of tested peptides (each 100 MM). [0028] FIG. 1C shoWs the level of cyclin E/CDK2 complex formation and CaM-cyclin E interaction in the presence of each peptide (100 uM) by co-immunoprecipitation analysis. [0029] FIG. 1D shoWs a histone H1 in vitro kinase assay from Gl/S-synchroniZed mouse VSMC With differing Ca2+ concentrations in the presence of CBS (SEQ ID NO:2) or NC (both 100 MM). [0030] FIG. 2 shoWs the effects of the CBS peptide (SEQ ID NO:2) on VSMC proliferation. [0031] FIG. 2A shoWs confocal microscopy images (60x) of mouse VSMC after nucleofection of FITC tagged-CBS or -NC (both 100 MM). Green: FITC, Blue: Hoechst. [0032] FIG. 2B shoWs the level of proliferation of primary mouse aortic SMC (MTT assay) in the presence of CBS (SEQ ID NO:2) or NC peptides. [0033] FIG. 3 shoWs the mechanism of CBS-induced (SEQ ID NO:2) proliferation reduction. [0034] FIG. 3A shoWs a Lactate dehydrogenase (LDH) assay after CBS (SEQ ID NO:2) and NC peptide delivery (1 mM) into asynchroniZed primary mouse aortic SMC.

US 2011/0105382 A1

[0035] FIG. 3B depicts the cell cycle analysis after delivery of CBS (SEQ ID N012) or NC peptides (1 mM) into starved (GO-phase cell cycle synchronized) and then serum-stimu lated primary mouse aortic SMC. [0036] FIG. 3C shoWs Western blotting after delivery of the CBS (SEQ ID N012) or NC peptides. Representative blots from three separate experiments are shoWn With normalized band intensities. [0037] FIG. 3D shoWs a MTT cell proliferation assay com paring the CBS (SEQ ID N012), scramble peptide control (sequence: FAFGRQVNKARSEKALGVSDRT (SEQ ID N016)) and NC peptide. All peptides Were nucleofected at the same concentration of 1 mM. *P<0.05. [0038] FIG. 4 shoWs that CBS reduces the proliferation of VSMC in a cyclin E-speci?c/dependent manner. [0039] FIG. 4A shoWs Western blotting from Wild type (WT) and cyclin E1/E2 double knock out- (K0) mouse embryonic ?broblasts (MEFs). [0040] FIG. 4B is a MTT assay showing the level of pro liferation of WT- & K0-MEFs in the presence (1 mM) or absence of peptide delivered into asynchronized cells. *P<0. 05. [0041] FIG. 4C reveals the cell cycle analysis of WT- & K0-MEFs after treatment of calmidazolium (CMZ, nonse lective CaM inhibitor) or peptides (CBS (SEQ ID N012) or NC). Each treatment Was performed on starved cells folloWed by serum stimulation With 10% FBS for 20 hr (for CMZ treatment) or 24 hr (for peptide treatment via nucleofection). *P<0.05, **P<0.01 [0042] FIG. 5 shoWs the results of treatment of TAT-CBS (SEQ ID N012)-His peptide to VSMC. [0043] FIG. 5A shoWs the sequence of TAT-CBS (SEQ ID N012)-His peptide. 10 amino acids from the TAT domain of HIV-1 Were fused to the N-terminus of CBS (SEQ ID N012). 6><-His tag Was also fused at the C-terminus. [0044] FIG. 5B shoWs Western blotting after TAT-CBS-His or TAT-NC-His peptide treatment (both 100 uM) to primary mouse aortic SMC. Peptides Were added to serum free DMEM cell culture media for 1 hr. [0045] FIG. 5C is a MTT assay shoWing the level of pro liferation of primary mouse aortic SMC in the presence TAT CBS (SEQ ID N012) and TAT-NC (both 100 uM). Peptides Were added to serum free DMEM cell culture media for 1 hr folloWed by fresh media replacement With 10% FBS. MIT assay Was performed after 2 days. Relative 0D intensity Was obtained by normalizing each intensity to control group With out peptide. **P<0.01. [0046] FIG. 5D shoWs the dose-response curve and IC5O measurement of TAT-CBS (SEQ ID N012) for the inhibitory effects on primary VSMC proliferation. MTT assay Was per formed 2 days after peptide treatment. An IC5O value Was calculated by nonlinear regression analysis based on a Bolt zmann sigmoid curve from 3 separate experiments. [0047] FIG. 5E shoWs the proliferation of three human cancer cellsiHeLa (human cervical cancer cell), Saos-2 (hu man osteosarcoma), and A549 (human lung cancer cell)iin the presence TAT-CBS-His (100 uM). Relative 0D intensity Was obtained by normalizing each intensity to control group Without peptide. **P<0.01 vs. No pep. [0048] FIG. 6 depicts an in vivo application of TAT-CBS to a mouse carotid artery injury model. [0049] FIG. 6A shoWs images (10x) of H&E stained injured and uninjured mouse carotid arteries With and Without treatment of TAT-CBS (SEQ ID N012)-His or TAT-NC-His.

May 5,2011

[0050] FIG. 6B shoWs media mass (umz) and Intima/ Media (UM) ratios of mouse common carotid arteries harvested at 14 days folloWing injury. All injuries Were performed on the right side, While the left common carotid artery of each mouse Was used as an uninjured control. *P<0.05, **P<0.01

[0051] FIG. 6C shoWs representative images of PCNA staining (20x). [0052] FIG. 6D shoWs the percentage of PCNA-positive nuclei at 14 days folloWing injury. **P<0.01 [0053] FIG. 7 shoWs a small motif in CBS.

[0054] FIG. 7A is a MTT assay With TAT-truncated pep tides With primary mouse aortic SMC, and human cancer cells HeLa, Saos-2, or A549. All peptides Were employed at the concentration of 100 [1M, and the MTT assay Was per formed 2 days later. The sequence of each peptide is shoWn in Table 1A.

[0055] FIG. 7B is a histone H1 kinase assay With 7 mer peptides. Each peptide (200 uM) Was added during the immu noprecipitation step With cyclin E antibody. Sequence of each peptide is shoWn in Table 1B. *P<0.05 vs. No pep.

[0056] FIGS. 7C & D shoWs MTT assays With additional TAT-truncated peptides With primary mouse aortic SMC, and human cancer cells HeLa, Saos-2 or A549. All peptides Were employed at the concentration of 100 [1M, and the MTT assay Was performed 2 days later. The sequence of each peptide is shoWn in Table 1C, and 1D, respectively. [0057] FIG. 8 shoWs an in vivo application of truncated peptides to mouse carotid artery injury model.

[0058] FIG. 8A shoWs media mass (umz) and FIG. 8B shoWs Intima/Media (I/M) ratios of mouse common carotid arteries harvested at 14 days folloWing injury. All injuries Were performed on the right side, While the left common carotid artery of each mouse Was used as an uninjured control.

*P<0.05, **P<0.01 [0059] FIG. 9 shoWs the isolated nucleic acid sequence of SEQ ID N01 1 and the amino acid sequence of SEQ ID N012, SEQ ID N01 3, SEQ ID N01 4, and SEQ ID N01 5. Option ally, SEQ ID N012 is obtained from human, SEQ ID N01 3 is obtained from mouse, SEQ ID N01 4 is obtained from rat, and SEQ ID N01 5 is obtained from chicken.

[0060] FIG. 10 shoWs the anti-proliferative effect of TAT CBS using tritiated-thymidine assays in primary human aor tic VSMC (FIG. 10A), MEF (FIG. 10B) and Cyclin E1/2 K0 MEF (FIG. 10C) treated With 10 and 100 [1M of TAT-CBS, TAT-NC or TAT-Scramble for 1.0 h at 37° C. prior to the start of the thymidine incorporation assay. Compared to controls, TAT-CBS inhibited 3H-thymidine incorporation in primary human aortic SMC in vitro

[0061] FIG. 10A shoWs a dose-dependent, anti-prolifera tive effect of TAT-CBS 72 h after treatment in human aortic VSMC compared to control cells treated With TAT-NC (n:3, each condition Was done in triplicate and average values are reported). *P<0.05 [0062] FIG. 10B shoWs a dose-dependent, anti-prolifera tive effect of TAT-CBS 48 h after treatment in MEF (n:2, each condition Was done in triplicate and average values are reported). **P<0.01 [0063] FIG. 10C shoWs that in Cyclin E1/E2 K0 MEF cells, TAT-CBS is not able to produce an anti-proliferative effect 48 h after treatment. Proliferation values are similar to

US 2011/0105382 A1

untreated cells and negative controls (n:2, each condition Was done in triplicate and average values are reported).

DETAILED DESCRIPTION OF THE INVENTION

[0064] The invention relates to isolated peptides useful in treatment of cancer by reducing cancer cell proliferation. The peptides are also useful in treatment of vascular disease by reducing smooth muscle cell proliferation. The peptides are useful because they disrupt calmodulin binding to cyclin E protein in order to inhibit CDK2 activity. [0065] In particular, the invention relates to amino acid sequences, termed ‘CBS’-Calmodulin Binding Sequence, that (i) inhibit the binding of CaM to cyclin E, (ii) abrogate Ca2+-sensitive cyclin E/CDK2 activity, (iii) block G1 to S cell cycle progression of vascular smooth muscle cells (“VSMC”) and (iv) reduce their rate of proliferation in vitro. The CBS peptides (v) inhibit the knoWn ‘activating’ pho spho rylation of CDK2 on Thr160 (65% inhibition) by selectively inhibiting CaM-cyclin E interactions. The effect is (vi) selec tive because the binding of CaM to another target protein, calcineurin, Was not altered by the CBS peptide, and (vii) both the Ca2+-sensitive CDK2 activity and (viii) proliferation of mouse embryonic ?broblasts de?cient in cyclins E (cyclin E1/E2 double knock out mice are embryonic lethal) Were not inhibited by the CBS peptide. Finally, (ix) the anti-prolifera tive effect of the CBS peptides are similar betWeen VSMC (IC50:8.89:1.24 and several human cancer cell lines, such as HeLa (cervical cancer cells), Saos-2 (osteosarcoma), and A549 (lung cancer), and (x) occurs in the absence of cytotox icity. CBS peptides inhibit (a) cyclin E-speci?c, Ca2+/CaM dependent, CDK2 activity, and (b) Ca2+-sensitive cell cycle progression and cell proliferation With high target and mecha nistic speci?cities respectively in both VSMC and human cancer cell lines in vitro. Inhibitors of CaM-cyclin E interac tions are therefore useful as therapeutics for diseases charac teriZed by uncontrolled cell proliferation. [0066] The peptides of the invention have “CBS activity” Which can be readily assessed With an assay measuring cell proliferation levels, such as an MIT assay. If a peptide of the invention reduces VSMC or cancer cell proliferation in a statistically signi?cant manner, then it has CBS activity. For example, a peptide having CBS activity optionally reduces proliferation activity by providing at least 50% or at least 70% inhibition effect on the proliferation of VSMC and the cancer cell lines tested in FIG. SE. [0067] The isolated CBS peptides are useful for treating a number of diseases. In one embodiment, the invention includes methods of reducing cell proliferation caused by cyclin E-speci?c calcium/calmodulin-dependent CDK2 activity in a cell, by administering a CBS peptide to the cell. [0068] The invention also includes methods of treating can cer in a mammal by administering a CBS peptide to the mammal. Examples of cancer include cervical cancer, osteosarcoma cancer or lung cancer.

[0069] In another embodiment, a CBS peptide is adminis tered to a mammal in a method of reducing proliferation of vascular smooth muscle cells. This effect has been demon strated in vivo in arteries. Histological examination, mor phometry, and immunohistochemical staining for the prolif erating cell nuclear antigen (PCNA) of arteries harvested 2 Weeks after injury by Wire denudation revealed that TAT-CBS (SEQ ID NO:2)-His treatment signi?cantly decreased the vascular smooth muscle cell proliferative response to injury as manifested by reduced medial mass, intima:media ratios,

May 5,2011

and PCNA-positive nuclei in TAT-CBS(SEQ ID NO:2)-His vs. F-127 only- and TAT-NC-His-treated groups (FIG. 6) [0070] In another embodiment, the method involves administration of a CBS peptide for treatment of a vaso occlusive disorder. [0071] The invention also includes an isolated nucleic acid encoding a CBS peptide of the invention. The invention fur ther includes an isolated antibody selectively binding a CBS peptide.

Peptides of the Invention

[0072] Isolated peptides comprising all or part of any of SEQ ID NO:2 and having cell proliferation inhibition activity are examples of useful peptides (FIG. 9). SEQ ID NO:2 corresponds to the amino acid sequence for the calmodulin (CaM) binding region of human cyclin E1. In alternate embodiments, the invention includes isolated peptides based on homologous 22 amino acid sequences or fragments thereof from other animals, such as mammals and birds. For example, FIG. 9 shoWs 22 amino acid sequences from rat, mouse and chicken that have CBS activity (SEQ ID NOS:3 to 5). The isolated peptides of the invention are typically 50 amino acids or less, optionally less than: 35, 30, 25, 20, 15, 10. 9, 8, 7 or 6 amino acids long. [0073] In other embodiments, the inventors have shoWn that peptides as small as 5 amino acids of one of the foregoing sequences have CBS activity and are useful to treat cancer and vascular disease, for example:

Human: VTVFL

Mouse: VAVFL

Rat: VPVFL

Chicken: VATFL

[0074] Optionally, the isolated peptide comprises at least: 5, 6, 7, 8, 9, 10, 11, 15 or 18 amino acids ofSEQ ID NO:2-5. Optionally, the peptide comprises a fragment of 5-10, 10-15, 15-20 or 20-21 amino acids ofa peptide of SEQ ID NO:2-5 (FIG. 9), Wherein the peptide reduces cell proliferation. Examples of useful fragments based on SEQ ID NO:2 include amino acid sequences selected from the group consisting of (amino acids 2-21 of SEQ ID NO:2, amino acids 3-20 of SEQ ID NO: 2, amino acids 4-19 of SEQ ID NO: 2, amino acids 5-18 of SEQ ID NO: 2, amino acids 6-17 of SEQ ID NO: 2, amino acids 7-16 ofSEQ ID NO: 2, amino acids 8-15 ofSEQ ID NO: 2, amino acids 9-14 of SEQ ID NO: 2, amino acids 12-20 ofSEQ ID NO: 2, amino acids 14-20 ofSEQ ID NO: 2, amino acids 16-20 of SEQ ID NO: 2). [0075] Particularly useful fragments include peptides hav ing a sequence V-X1-X2-F-L, Wherein X1 is optionally a hydrophobic amino acid or a neutral amino acid (eg. selected from the group ofV, I, L, F, W, C, A,Y, H, T, S, P, G, R or K), typically T, A, P and Wherein X2 is optionally a hydrophobic amino acid or a neutral amino acid (eg. selected from the group ofV, I, L, F, W, C, A, Y, H, T, S, P, G, R or K), typically V or T.

[0076] Longer peptide fragments including these 5 mer sequences are also useful, for example, peptides including 5, 6, 7, 8, 9, 10, 11, 15, 18 or 22 amino acids (such as peptides based on SEQ ID NO:2 shoWn in FIG. 9). [0077] Typically, fragments Will comprise three consecu tive hydrophobic residues located Within 5 amino acids of the C-terminus, such as VFL of SEQ ID NO:2. Optionally, at

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CBS patent_US20110105382