Calmodulin-Binding Peptides for Treating Cancer and Smooth Muscle Proliferation

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 onAug.
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 ofat 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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GGAEFSARSRKRKANVTVFLQD SEQ lD NO; 2 (human)
GGSDLSVRSRKRKANVAVFLQD SEQ ID NO: 3 (mouse)
GGSDLS'VRSRKRKPNVPVFLQD SEQ ID NO: 4 (rat)
GGAEGTVRARKRKADVATFLQD SEQ ID NO: 5 (chicken)

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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 foruse 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 causedby
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 ofa type ofchemotherapy 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 ofballoon 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 ofCaM as a regulator ofcell cycle progression
has also been recognized. A direct interaction betWeen the
major calcium {Ca2+} signal-transducing protein calmodu
lin (CaM), and the critical regulator ofcell 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 shorterpeptides 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 ofthe inventionreduce 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 ofthe C-terminus. Optionally, at least one ofthe 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 ofthe invention, the peptide comprises
a truncated amino acid sequence ofthe 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 ofthe 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 ofthe 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 ofthe invention may include
various structural forms of the primary CBS peptide Which
retainbiological activity. For example, a peptide ofthe 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 ofthe 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.
[0016] The invention furtherrelates to the use ofthe peptide
or the pharmaceutical composition ofthe 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 thereofWherein 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 ofcalmodulin to cyclin E protein.
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
thereofWherein 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
brovasculardisease. The hypertension optionally is causedby
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 proteinmediated cell proliferation.
Optionally, the pharmaceutical composition is a stent.
tides or the pharmaceutical composition for the treatment ofa
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 causedbyvascularinjury. Optionally, the
disorder comprises vascular smooth muscle cells undergoing
calcium sensitive cyclin E proteinmediated 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 ofthe peptides or the pharmaceutical composition ofthe
invention.
[0023] Other features and advantages ofthe 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 ofthe 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 ofthe 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 ofcyclin 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 ofCBS (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)
ofmouse VSMC after nucleofection ofFITC tagged-CBS or
-NC (both 100 MM). Green: FITC, Blue: Hoechst.
[0032] FIG. 2B shoWs the level ofproliferation of primary
mouse aortic SMC (MTT assay) inthe presence ofCBS (SEQ
ID NO:2) or NC peptides.
[0033] FIG. 3 shoWs the mechanism ofCBS-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
ofCBS (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 ofthe
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 ofpeptide 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 ofCBS (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 ofprimary 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
obtainedby normalizing each intensity to control group With
out peptide. **P<0.01.
[0046] FIG. 5D shoWs the dose-response curve and IC5O
measurement ofTAT-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 ofTAT-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 ofeachmouse
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 ofeach peptide is shoWn in
Table 1A.
[0055] FIG. 7B is a histone H1 kinase assay With 7 mer
peptides. Eachpeptide (200 uM) Was added duringthe immu
noprecipitation step With cyclin E antibody. Sequence ofeach
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 ofeachmouse 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 ofSEQ 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
ofthe 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 ofTAT-CBS 48 h aftertreatment 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 ofcancer 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 ofproliferation in vitro.
The CBS peptides (v) inhibit the knoWn ‘activating’ phospho
rylation ofCDK2 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 ofcytotox
icity. CBS peptides inhibit (a) cyclin E-speci?c, Ca2+/CaM
dependent, CDK2 activity, and (b) Ca2+-sensitive cell cycle
progression andcell proliferationWithhightarget andmecha
nistic speci?cities respectively in both VSMC and human
cancer cell lines in vitro. Inhibitors ofCaM-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 ofVSMC 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 oftreating 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 andhaving 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 orless, 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 ofone ofthe 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 of5-10, 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 ofuseful fragments based on SEQ ID NO:2 include
amino acid sequences selected from the group consisting of
(amino acids 2-21 ofSEQ ID NO:2, amino acids 3-20 ofSEQ
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 ofthe
C-terminus, such as VFL of SEQ ID NO:2. Optionally, at

Calmodulin-Binding Peptides for Treating Cancer and Smooth Muscle Proliferation

Calmodulin-Binding Peptides for Treating Cancer and Smooth Muscle Proliferation

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