U.S. patent application number 16/810667 was filed with the patent office on 2020-08-27 for treatment of damaged nerve with pten inhibitor.
This patent application is currently assigned to Kolon TissueGene, Inc.. The applicant listed for this patent is Kolon TissueGene, Inc.. Invention is credited to Kwangwook AHN, Kwan Hee LEE, Moon Jong NOH.
Application Number | 20200270319 16/810667 |
Document ID | / |
Family ID | 1000004824498 |
Filed Date | 2020-08-27 |
![](/patent/app/20200270319/US20200270319A1-20200827-D00001.png)
![](/patent/app/20200270319/US20200270319A1-20200827-D00002.png)
![](/patent/app/20200270319/US20200270319A1-20200827-D00003.png)
![](/patent/app/20200270319/US20200270319A1-20200827-D00004.png)
![](/patent/app/20200270319/US20200270319A1-20200827-D00005.png)
United States Patent
Application |
20200270319 |
Kind Code |
A1 |
LEE; Kwan Hee ; et
al. |
August 27, 2020 |
TREATMENT OF DAMAGED NERVE WITH PTEN INHIBITOR
Abstract
The present application discloses a method of growing or
proliferating nerve cells by contacting the cells with phosphatase
and tensin homolog (PTEN) lipid phosphatase inhibiting peptide.
Inventors: |
LEE; Kwan Hee; (Rockville,
MD) ; NOH; Moon Jong; (Rockville, MD) ; AHN;
Kwangwook; (Rockville, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Kolon TissueGene, Inc. |
Rockville |
MD |
US |
|
|
Assignee: |
Kolon TissueGene, Inc.
Rockville
MD
|
Family ID: |
1000004824498 |
Appl. No.: |
16/810667 |
Filed: |
March 5, 2020 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15142324 |
Apr 29, 2016 |
10584153 |
|
|
16810667 |
|
|
|
|
PCT/US2014/063900 |
Nov 4, 2014 |
|
|
|
15142324 |
|
|
|
|
61899795 |
Nov 4, 2013 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 9/16 20130101; A61K
38/00 20130101; C07K 14/4703 20130101; C12Y 301/03067 20130101 |
International
Class: |
C07K 14/47 20060101
C07K014/47; C12N 9/16 20060101 C12N009/16 |
Claims
1. A method of regenerating nerve or attenuating degeneration of
nerve at a site of nerve injury comprising administering at or an
area near an injured nerve, a nerve regenerating or nerve
degeneration attenuating amount of phosphatase and tensin homolog
(PTEN) lipid phosphatase inhibiting peptide.
2. The method according to claim 1, wherein the PTEN inhibitor
peptide is modified PTEN peptide or fragment thereof in which
phosphorylation site is modified such that a serine or threonine in
the phosphorylation site is phosphorylated.
3. The method according to claim 1, wherein the phosphorylated
serine or threonine is located at position Thr-366, Ser-370,
Ser-380, Thr-382, Thr-383 or Ser-385.
4. The method according to claim 1, wherein the phosphorylated
serine or threonine is located at position Ser-370, Ser-380 and/or
Ser-385.
5. The method according to claim 3, wherein the phosphorylated
serine or threonine is located at position Ser-370, Ser-380 and
Ser-385.
6. The method according to claim 3, wherein the phosphorylated
serine or threonine is located at position Ser-380 and Ser-385.
7. The method according to claim 1, wherein the peptide is a
fragment of a peptide of phosphorylation site and/or PDZ domain
binding motif.
8. The method according to claim 1, wherein the peptide further
comprises a peptide transfer domain (PTD).
9. The method according to claim 1, wherein the nerve injury is in
the central nervous system.
10. A peptide which inhibits phosphatase and tensin homolog (PTEN)
lipid phosphatase activity.
11. The peptide according to claim 10, wherein the peptide is a
PTEN peptide or fragment thereof in which phosphorylation site is
modified such that a serine or threonine in the phosphorylation
site is phosphorylated.
12. The peptide according to claim 11, wherein the phosphorylated
serine or threonine is located at position Thr-366, Ser-370,
Ser-380, Thr-382, Thr-383 or Ser-385.
13. The peptide according to claim 12, wherein the phosphorylated
serine or threonine is located at position Ser-370, Ser-380 and/or
Ser-385.
14. The peptide according to claim 13, wherein the phosphorylated
serine or threonine is located at position Ser-370, Ser-380 and
Ser-385.
15. The peptide according to claim 13, wherein the phosphorylated
serine or threonine is located at position Ser-380 and Ser-385.
16. The peptide according to claim 11, wherein the peptide is a
fragment of a peptide of phosphorylation site and/or PDZ domain
binding motif.
17. The peptide according to claim 11, wherein the peptide further
comprises a peptide transfer domain (PTD).
18. A method of growing, proliferating or enhancing cell activity
of a nerve cell comprising contacting the nerve cell with a
phosphatase and tensin homolog (PTEN) lipid phosphatase inhibiting
peptide.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
[0001] The present application relates to a method of regenerating
nerve or attenuating degeneration of injured nerve by administering
at or an area near an injured nerve, a nerve regenerating or nerve
degeneration attenuating amount of phosphatase and tensin homolog
(PTEN) lipid phosphatase inhibiting peptide.
2. General Background and State of the Art
[0002] In adult mammalian nervous system, regeneration of damaged
neurons hardly occurs in healing response to nerve injury. There
are two main reasons why adult CNS neurons fail to regenerate after
injury--axons do not regenerate in adult central nervous system not
only because of its inhibition by secreted extracellular inhibitory
factors upon injury, but also because of the loss of intrinsic axon
growth ability, which rapidly declines through aging [Schwab et al;
1996, Goldberg et al. 2002; Filbin et al. 2006; Fitch et. al 2008].
However, elimination of extracellular inhibitory molecules secreted
upon nerve injury only triggers very limited axon regeneration in
vivo [Yiu et. al 2006; Hellal et al. 2011]. Thus, promoting axonal
regeneration process by regulation of intrinsic nerve outgrowth is
currently focus of a therapeutic target for nerve injury
treatment.
[0003] PTEN (phosphatase and tensin homolog) protein is a dual
phosphatase and is considered to be important as tumor suppressor
by negatively regulating phosphatidylinositol3-kinase (PI3K)
signaling pathway. The PI3K signaling pathway is a critical signal
transduction pathway for cell proliferation, survival and
differentiation as well as protein synthesis, metabolism and
motility [Zhang et al. 2010]. As a lipid phosphatase, PTEN
catalyzes conversion of phosphatidylinositol (3,4,5) triphosphate
(PIP3) to phosphatidylinositol (4,5) diphosphate (PIP2) by
dephosphorylating the 3-position of PIP3, hence suppressing PI3K
signaling pathway by antagonizing PI3K activity. [Di Cristofano et.
al 2010]. Deletion or inactivation of PTEN enhances PI3K activity
and promotes activation of downstream components of PI3K signaling
pathway, including PDK1, Akt and mammalian target of rapamycin
(mTOR), which leads to tumor formation [Di Cristofano et. al 2010;
Stambolic et al. 1998].
[0004] Regulation of PI3K-mediated signaling by PTEN is also deeply
related to nerve regeneration process in nerve system. Recent
studies reveal that inhibition of PTEN protein or deletion of PTEN
gene facilitates intrinsic regenerative outgrowth of adult CNS/PNS
nerve upon Injury [Park et. al 2008; Liu et. al 2010; Sun et. al
2012; Christie et. al 2012]. For example, Park et al. found that
deletion of PTEN in adult rat retinal ganglion cells (RGCs) using
conditional knockout mice actually promotes robust axon
regeneration after optic nerve injury by re-activating
PI3K-Akt-mTOR signaling pathway. Reactivating mTOR pathway by
conditional knockout of another negative regulator of the mTOR
pathway also leads to axon regeneration, indicating that promotion
of PI3K-mTOR signaling may be a key factor for restoring intrinsic
axon regeneration ability. Also, Liu et al. reported that
conditional deletion of PTEN in in vivo CNS injury model actually
increases the diminished neuronal mTOR activity upon CNS injury by
up-regulating PI3K signaling pathway, which leads to enhanced
compensatory sprouting of uninjured CST axons and successful
regeneration of injured CST axons past a spinal cord lesion. In
case of PNS injury, inhibition of PTEN both in vitro and in vivo
also increases axonal outgrowth [Christie et. al 2012]. Thus,
developing PTEN inhibitor for promoting PI3K-mTOR signaling pathway
is a good therapeutic target to enhance axon regeneration in
injured nerve system. the PTEN inhibitor may be used in combined
therapeutic methodology with existing or novel cell therapy
containing other effective reagents for nerve regeneration after
CNS or PNS injury.
[0005] In this study, we developed potential PTEN inhibitors
effective for nerve regeneration and/or protection from nerve
degeneration by stimulating PI3K signaling pathway. For activation
of PTEN as lipid phosphatase, PTEN must localize in the plasma
membrane in an appropriate orientation [Leslie et. al 2008]. Thus,
we investigated the mechanism of PTEN membrane localization to
design potential PTEN inhibitor candidates in peptide form. Three
different peptides--TGN-1, TGN-2 and TGN-3--were designed and
synthesized as potential PTEN inhibitors and their inhibitory
ability against PTEN activity using in vitro PTEN activity assay
was investigated. We also characterized their effect on regulation
of PI3K signaling pathway by using neuronal cell lines. We
discovered that TGN-1 and TGN-2 peptides, which are modified
peptides mimicking the phosphorylation site in PTEN C-terminal
region, actually diminished PTEN lipid phosphatase activity in in
vitro PTEN activity assay. TGN-1 peptide also enhanced the
activation level of Akt protein in PC12 cells, indicating that
these peptides are effective to up-regulate PI3K-Akt signaling
pathway. Neurite assay with neuronal cell showed that TGN-1 and
TGN-2 peptides promoted neurite outgrowth as well as delayed
neurite degeneration by enhancing neurite microtubule structure.
Therefore, TGN peptides are useful as a therapeutic agent for nerve
regeneration after CNS injury.
SUMMARY OF THE INVENTION
[0006] In one aspect, present invention is directed to the
following:
[0007] In one aspect, the invention is directed to a method of
regenerating nerve or attenuating degeneration of nerve at a site
of nerve injury comprising administering at or an area near an
injured nerve, a nerve regenerating or nerve degeneration
attenuating amount of phosphatase and tensin homolog (PTEN) lipid
phosphatase inhibiting peptide. The PTEN inhibitor peptide may be
modified PTEN peptide or fragment thereof in which phosphorylation
site is modified such that a serine or threonine in the
phosphorylation site is phosphorylated. The phosphorylated serine
or threonine may be located at position Thr-366, Ser-370, Ser-380,
Thr-382, Thr-383 or Ser-385. The phosphorylated serine or threonine
may be located at position Ser-370, Ser-380 and/or Ser-385. The
phosphorylated serine or threonine may be located at position
Ser-370, Ser-380 and Ser-385. The phosphorylated serine or
threonine may be located at position Ser-380 and Ser-385. The
peptide may be a fragment of a peptide of phosphorylation site
and/or PDZ domain binding motif. The peptide may further comprise a
peptide transfer domain (PTD). The nerve injury may be in the
central nervous system.
[0008] In another aspect, the invention is directed to peptide
which inhibits phosphatase and tensin homolog (PTEN) lipid
phosphatase activity. The PTEN inhibitor peptide may be modified
PTEN peptide or fragment thereof in which phosphorylation site is
modified such that a serine or threonine in the phosphorylation
site is phosphorylated. The phosphorylated serine or threonine may
be located at position Thr-366, Ser-370, Ser-380, Thr-382, Thr-383
or Ser-385. The phosphorylated serine or threonine may be located
at position Ser-370, Ser-380 and/or Ser-385. The phosphorylated
serine or threonine may be located at position Ser-370, Ser-380 and
Ser-385. The phosphorylated serine or threonine may be located at
position Ser-380 and Ser-385. The peptide may be a fragment of a
peptide of phosphorylation site and/or PDZ domain binding motif.
The peptide may further comprise a peptide transfer domain (PTD).
The nerve injury may be in the central nervous system.
[0009] In yet another aspect, the invention is directed to a method
of growing, proliferating or enhancing activity of a nerve cell
comprising contacting the nerve cell with tensin homolog (PTEN)
lipid phosphatase inhibiting peptide.
[0010] These and other objects of the invention will be more fully
understood from the following description of the invention, the
referenced drawings attached hereto and the claims appended
hereto.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] The present invention will become more fully understood from
the detailed description given herein below, and the accompanying
drawings which are given by way of illustration only, and thus are
not limitative of the present invention, and wherein;
[0012] FIGS. 1A-1B show design of TGN peptides as potential PTEN
inhibitor. FIG. 1A) Diagram of PTEN C-terminal Region. PTEN
C-terminal region include C2 domain (AA186.about.403),
phosphorylation site (AA352.about.399) and PDZ domain binding motif
(400.about.403). The phosphorylation site and PDZ domain binding
motif containing region (AA352.about.403) were used as template for
TGN peptide design. FIG. 1B) Amino acid sequence of TGN peptides.
TGN-1, TGN-2 and TGN-3 peptides mimic PTEN phosphorylation site, in
which the indicated residues were modified by phosphorylation.
TGN-4 peptide is a scrambled peptide for TGN-1, and TGN-5 peptide
is a scrambled peptide for TGN-2.
[0013] FIGS. 2A-2C show In vitro PTEN Activity Assay with TGN
Peptides. FIG. 2A) Mechanism of In vitro PTEN Activity Assay using
Malachite Green Assay Kit. C8-PIP3 was used as PTEN substrate and
prepared as liposome with other phospholipids (DOPC and DOPC). The
phosphate ions produced by PTEN from C8-PIP3 were measured by
monitoring the optical density of phosphate ion-Malachite Green
reagent complex at 620 nm. FIG. 2B) Effect of TGN peptides against
in vitro PTEN activity. TGN-1, TGN-2 and TGN-3 peptides were
examined for their PTEN inhibitory effect via in vitro PTEN
activity assay. 10 .mu.M of each peptide was incubated with 20 ng
of human recombinant PTEN protein and 0.1 mM of C8-PIP3 as liposome
in 100 .mu.l, of reaction volume. TGN-4 and TGN-5 peptides were
used to check the sequence specificity for TGN-1 and TGN-2/3
peptides, respectively. All data represent results of
experimentation in triplicate. FIG. 2C) IC.sub.50 curves for TGN-1
and TGN-2 peptides. IC.sub.50 values were measured via in vitro
PTEN activity assay with TGN-1 and TGN-2 peptides in dose-dependent
manner and calculated via Prism 5 software. IC.sub.50 values for
TGN peptide are 19.93 .mu.M for TGN-1, 4.83 .mu.M for TGN-2 and
87.12 .mu.M for TGN-3.
[0014] FIGS. 3A-3C show that TGN-1 peptide promotes PI3K-Akt
signaling by increasing Akt activation level in vivo. FIG. 3A)
Mechanism of Akt activation by blocking PTEN activity using TGN-1.
Introduction of TGN-1 in PI3K signaling pathway facilitates PI3K
signaling and promotes Akt activation (phosphorylation) level. FIG.
3B) Western blot data with PC12 cell lysates. PC12 cells were
treated with either TGN-1 peptide (10 .mu.M, 100 .mu.M) or TGN-4
peptide (10 .mu.M) and incubated for 24 hr. Western blot data using
anti-phospho Akt antibody showed that TGN-1 specifically promotes
endogenous Akt activation level in dose-dependent manner. FIG. 3C)
The expression level of PTEN and .beta.-actin were also monitored
as positive and loading control.
[0015] FIGS. 4A-4B show TGN-1 and TGN-2 peptide that show
neurotrophic effects and neuroprotection effect against neurite
degeneration. FIG. 4A) Differentiated PC12 cells were firstly
treated with Nocodazole (0.5 .mu.M) for 1 hr, and incubated with
fresh media containing NGF (long/mL) and TGN peptides (TGN-1 and
TGN-2, 100 .mu.M/each) for additional 72 hrs. Relative neurite
stability was calculated as a ratio of green/red fluorescence
signal intensities from immunofluorescence images using Image J
software. All fluorescence signal intensities were measured at
least 3 times per each sample for green/red ratio calculation and
normalized (media only=100%). FIG. 4B) Quantification of neurite
outgrowth on differentiated PC12 cells. PC12 cells were treated
with differentiation medium containing NGF (50 ng/ml) for 24 hr,
followed by incubation with TGN peptides (100 .mu.M/each) for
additional 2 days. TGN-4 peptide was used as a negative control for
TGN-1. Neurite quantification was performed spectrophotometrically
using neurite quantification kit (Millipore) at day 3 and
normalized (Media only=100%).
[0016] FIG. 5 shows a hypothetical model of the interfacial
activation of PTEN at cell membrane surface. PTEN 15 currently
believed to have two conformational states in vivo and is proposed
to undergo conformational change to localize in the membrane
localization in order to fully express its lipid phosphatase
activity. Soluble form of PTEN is in inactive state with "closed"
conformation, where the phosphorylated sites of PTEN C-terminal
region spatially mask PTEN active site and C2 domain to prevent
PTEN membrane association. When the phosphorylated residues in the
"phosphorylation site" are de-phosphorylated, PTEN changes its
conformation from "closed" conformation to "open" conformation. In
this stage, multiple membrane-binding motifs located at C2 domain
of PTEN are exposed and are ready to associate with a membrane. The
binding pocket of PTEN active site is also available for accessing
PIPS substrate residing on the membrane surface. Binding of PIP2 on
the membrane surface with N-terminal PIP2 binding motif as well as
the binding of C-terminal PDZ domain binding motif to PDZ domain in
adjutant protein (NHERF1) follow after PTEN is localized on the
cell membrane surface in its appropriate position required for its
lipid phosphatase activity to occur.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0017] In the present application, "a" and "an" are used to refer
to both single and a plurality of objects.
[0018] As used herein, injection of cells "near" an injured nerve
or neural system is meant that area which is close enough between
the injection site and the injury area to effect an efficacious
outcome of regenerating nerve or preventing degeneration of the
injured nerve cells at the injured site. Therefore, the injection
of cells at or near an injured nerve includes at the site of injury
or anywhere close enough for the injected cells to express the
effective polypeptide and the polypeptides are allowed to directly
or indirectly effect the nerve regeneration or nerve degeneration
preventing outcome. For peripheral nerve, especially in spinal cord
injury, the injection can be made "upstream" of the injury site
since cells tend to leak out at the site of injury.
[0019] As used herein, "neurite" refers to any projection from the
cell body of a neuron. This projection can be either an axon or a
dendrite. The term is frequently used when speaking of immature or
developing neurons, especially of cells in culture, because it can
be difficult to tell axons from dendrites before differentiation is
complete.
[0020] As used herein, "regeneration of nerve" means generation of
new nerve cells, neurons, glia, axons, myelins or synapses upon
nerve injury in either central nervous system (CNS) or peripheral
nervous system (PNS). The regeneration is driven by restored
intrinsic neuroregeneration ability via activation of
PI3K-mTOR-mediated signaling by inhibition of PTEN.
[0021] As used herein, "attenuation" or "prevention" of
degeneration of nerve means delaying the degeneration of axon, glia
or myelin stealth structure caused by nerve injury in either
central nervous system (CNS) or peripheral nervous system (PNS).
The "attenuation" or "prevention" is achieved by neuronal
microtubule structure stabilization closely related with
PI3K-mTOR-mediated signaling, which is activated by PTEN
inhibition.
[0022] Phosphatase and Tensin Homolog (PTEN)
[0023] PTEN amino acid sequence is as follows:
TABLE-US-00001 (SEQ ID NO: 1) 10 20 30 40 MTAIIKEIVS RNKRRYQEDG
FDLDLTYIYP NIIAMGFPAE 50 60 70 80 RLEGVYRNNI DDVVRFLDSK HKNHYKIYNL
CAERHYDTAK 90 100 110 120 FNCRVAQYPF EDHNPPQLEL IKPFCEDLDQ
WLSEDDNHVA 130 140 150 160 AIHCKAGKGR TGVMICAYLL HRGKFLKAQE
ALDFYGEVRT 170 180 190 200 RDKKGVTIPS QRRYVYYYSY LLKNHLDYRP
VALLFHKMMF 210 220 230 240 ETIPMFSGGT CNPQFVVCQL KVKIYSSNSG
PTRREDKFMY 250 260 270 280 FEFPQPLPVC GDIKVEFFHK QNKMLKKDKM
FHFWVNTFFI 290 300 310 320 PGPEETSEKV ENGSLCDQEI DSICSIERAD
NDKEYLVLTL 330 340 350 360 TKNDLDKANK DKANRYFSPN FKVKLYFTKT
VEEPSNPEAS 370 380 390 400 SSTSVTPDVS DNEPDHYRYS DTTDSDPENE
PFDEDQHTQI TKV
[0024] PTEN protein is currently becoming a popular target for
developing therapeutic material to regenerate injured nerve in
adult CNS system by restoring diminished intrinsic nerve
regeneration ability by promoting PI3K-Akt-mTOR signaling [Park et.
al 2008; Liu et. al 2010; Sun et. al 2012]. Development of novel
PTEN inhibitor is considered to be a good strategy for developing
PTEN-activity regulating molecules. Unfortunately, the X-ray
crystal structure of PTEN protein [Lee et. al 1999] is not
sufficient to provide enough information for PTEN-substrate (PIPS)
binding status, which is critical for designing effective PTEN
inhibitors directly blocking PTEN-substrate binding. Alternatively,
the mechanism by which PTEN targets the plasma membrane for its
activity is under intense investigation. Although the
phosphatidylinositol (3,4,5) diphosphate (PIP3), a substrate of
PTEN enzyme, is a member of phospholipids found in the cellular
membrane lipid bilayer, PTEN protein is originally produced as a
soluble protein and has to be activated interfacially for its lipid
phosphatase activity through conformational change, followed by
PTEN-membrane association in the proper orientation [Das et. al
2003; Leslie et. al 2008]. Several charged amino acids and binding
motifs located in PTEN C2 domain are considered to be the main
anchors to attach the PTEN protein on the cell membrane surface
[Lee et. al 1999; Georgescu et. al 2000; Leslie et. al 2008].
Additional binding using other binding moieties is also necessary
for PTEN to be properly orientated on the cellular membrane for
lipid phosphatase activity of PTEN to occur [Chambell et. al 2003;
Walker et. al 2004; Odriozola et. al 2007].
[0025] The unstructured part (AA 352-399) in the PTEN C-terminal
region is called "phosphorylation site" because this region
contains six Serine/Threonine (Thr-366, Ser-370, Ser-380, Thr-382,
Thr-383, and Ser-385) residues known as phosphorylation
modification sites [Lee et. al 1999; Vazquez et. al 2001]. Previous
studies revealed that mutation or deletion of these 6 residues in
this "phosphorylation site" leads to greater tumor suppressor
activity, enhanced PTEN membrane affinity, and reduced protein
stability [Vasquez et. al 2001; Das et. al 2003; Okahara et. al
2004; Randar et. al 2009].
[0026] Currently, it is believed that PTEN protein has two
conformation states (FIG. 4). In the "closed" conformation, PTEN is
inactive because the C-terminal region of PTEN including the
"phosphorylation site" masks membrane-binding motifs located in the
C2 domain as well as the PTEN active site pocket, preventing PTEN
association to cell membrane and PIP3 access to the active site. On
the other hand, PTEN becomes active interfacially in the "open"
conformation state, where the PTEN active site pocket and C2 domain
are both unmasked and totally exposed to cell membrane and its
substrate PIP3. Also, the phosphorylation state of these 6
Serine/Threonine residues in the "phosphorylation site" is
considered to be a critical factor for PTEN interfacial activation
because it directly controls conformational change of PTEN protein
from "closed" conformation to "open" conformation [Das et. al 2003,
Vasquez et. al 2006; Odriozola et. al 2007, Randar et. al
2009].
[0027] According to the currently suggested model (FIG. 5), there
are three steps required for the interfacial activation of PTEN at
a membrane surface.
[0028] 1) dephosphorylation of phosphorylated Serine/Threonine
residues in the "phosphorylation site" triggers PTEN conformational
change from "closed" to "open" conformation, which enables PTEN
protein to associate with cellular membrane and expose PTEN active
site pocket to PIP3 substrate located on the cell membrane.
[0029] 2) Multiple membrane-binding motifs in C2 domain then
interact with cell membrane to anchor PTEN protein on the membrane
surface.
[0030] 3) Additional Interaction between N-terminal PIP2 binding
site (AA6-15) and PIP2 molecule in the cellular membrane [Walker
et. al 2004] as well as the binding of C-terminal PDZ domain
binding site (AA400-403) with the PDZ domain of adjutant NHERF1
protein [Takahashi et. al 2006; Molina et. al 2010] are both also
required for adjustment of PTEN orientation on the cellular
membrane surface.
[0031] We designed our TGN peptide as potential PTEN inhibitor
based on the PTEN membrane localization model shown in FIG. 4, in
particular the "phosphorylation site" and PDZ domain-binding site
(AA 352-403). The basic concept of TGN peptide as potential PTEN
inhibitor is to prevent the association between PTEN and cell
membrane surface by masking PTEN active site and the C2 domain
required for membrane binding. As Ser370 and Ser385 are
preferentially phosphorylated via casein kinase II [Miller et. al
2002,] membrane localization as well as phosphatase activity are
increased, more than when other residues are mutated [Odriozola et.
al 2007]. Therefore at least one Serine residue out of these two
were included in all TGN peptides (Ser370/385 in TGN-1, Ser385 in
TGN-2/TGN-3). Also, phosphorylated Serine residues at 380 and 385
positions are currently considered to be part of
"pseudo-substrate", masking the catalytic pocket in PTEN active
site from accessing the real substrate PIP3 [Odriozola et. al
2007]. The peptides were designed to include these two Serine
residues (Ser 380 and Ser 385) in all of the TGN peptides.
[0032] TGN-1 peptide sequence mimics AA 365.about.388 region of
PTEN phosphorylation site and contains four Serine/Threonine
residues (Thr366, Ser370, Ser380 and Ser385) with three
phosphorylated modified residues (Ser370, Ser380 and Ser385). TGN-2
and TGN-3 peptide mimics AA376-403 region of PTEN protein,
including two phosphorylated Serine residues (Ser380 and Ser385) as
well as the C-terminal PDZ domain-binding motif (ITKV). Only the
Serine residues in both TGN-1 and TGN-2 peptides were
phosphorylated to mimic the phosphorylation site of PTEN in vivo
because phosphorylation of Threonine residues results in secondary
modification in vivo and is also less effective for altering
PTEN-membrane binding affinity when mutated [Odriozola et. al 2007;
Randar et. al 2009]. In TGN-3 peptide, two Serine residues (Ser380
and Ser385) were substituted with Valine for comparison.
Additionally, the sequences of TGN-1 and TGN-2/3 peptides were
scrambled to examine sequence specificity, and these peptides were
designated as TGN-4 and TGN-5 peptide, respectively.
[0033] In vitro activity assay and IC.sub.50 assay with recombinant
human PTEN protein and C8-PIP3 as substrate showed that TGN-1 and
TGN-2 peptides specifically inhibit PTEN activity in vitro in
dose-dependent manner (FIG. 2). C8-PIP3 was introduced to PTEN
protein as synthesized lipid vesicle--a mimicking system of cell
membrane lipid bilayer--with other phospholipid molecules
(DOPC/DOPS). The activity assay results implied that TGN-1 and
TGN-2 peptides may inhibit in vitro PTEN activity by directly
interacting with PTEN protein and interfering with PTEN-vesicle
membrane association to prevent the substrate (C8-PIP3) from
binding to the PTEN active site. In fact, in vitro PTEN activity
assay with direct addition of C-8 PIP3 lipid only instead of the
liposome form fails to show PTEN activity (data not shown). Much
reduced inhibitory effect by TGN-3 peptide compared with TGN-2
peptide suggests that phosphorylation modification on the Serine
residues (Ser380 and Ser385) is a significant factor for in vitro
PTEN inhibition by TGN-peptide. Also, TGN-2 peptide showed nearly
4-fold higher inhibitory effect on in vitro PTEN activity than
TGN-1 peptide (IC.sub.50 value for TGN-1 is 19.93 .mu.M and for
TGN-2 is 4.83 .mu.M). The main difference in structure between
TGN-1 and TGN-2 peptides is that the TGN-2 peptide contains the
last 15 amino acid sequence of PTEN C-terminal region
(AA389.about.403) including PDZ domain binding motif (AA
399.about.403). Since the activity assay was performed in in vitro
conditions, It may be explained that the last 15 amino acid
sequence present in TGN-2 peptide either provides higher binding
affinity toward PTEN protein to interfere with PTEN-vesicle
membrane association more efficiently or masks the substrate
binding pocket in PTEN active site more effectively than TGN-1
peptide.
[0034] TGN-1 peptide is also effective in blocking PTEN activity to
regulate PI3K-Akt signaling pathway in neuronal cells (FIG. 3).
PC12 cells containing endogenous or overexpressed PTEN were
incubated with TGN-1 for 24 hr and the activation (phosphorylation)
level of Akt protein was examined by Western blotting using
anti-phospho Akt antibody. The phosphorylation level of Akt protein
in cell lysates treated with TGN-1 peptide was much higher than the
lysates treated with TGN-4 peptide or DMSO, indicating that TGN-1
peptide specifically inhibits PTEN to antagonize PI3K activity.
Thus, TGN-1 peptide is effective in promoting PI3K-Akt signaling
pathway by suppressing PTEN activity.
[0035] Since microtubule stabilization is considered to be critical
for treating spinal cord injury by promoting axonal regeneration
ability and neuronal polarization [Sengottuvel et al 2011, Hellal
et al 2011, Witte et al 2008], we adopted Nocodazole to induce
neuritic degeneration on differentiated neuronal cells and tested
if TGN peptides show neuroprotective effect via microtubule
stabilization. As microtubule stability is closely related to
.alpha.-tubulin acetylation level [Takemura et al 1992], we
immunostained stable neurites with anti-acetylated .alpha.-tubulin
antibody. Immunofluorescence data (FIG. 4A) demonstrated that TGN-1
and TGN-2 peptides actually stabilized neurite microtubule
structure to delay neurite degeneration. Moreover, addition of
TGN-1 peptide specifically promotes neurite outgrowth on neuronal
cell differentiation process (FIG. 4B). Thus, TGN-1 and TGN-2
peptides show neurotrophic effect as well as neuroprotection
against neurite degeneration.
[0036] In a previous study, Odriozola et. al reported that
synthetic phosphomimic peptides (Cp-23, Cp-23DE) encompassing the
PTEN C-terminal phosphorylation site cluster (AA368.about.390),
similar to TGN-1 peptide sequence, mediates the suppression of PTEN
catalytic activity in vitro. Also, assays with 293T cells
transfected with GFP-fused phosphomimic peptides were shown to
decrease level of PTEN-membrane association and improve phospho-Akt
levels. However, the phosphomimic peptides (Cp-23, Cp-23DE) used in
Odriozola et al. mimics only the AA 368.about.390 region of PTEN
"phosphorylation site" but contains no phosphorylated Serine
residues as in the present TGN peptides. In fact, although the
Odriozola peptide (Cp23) and TGN-1 peptide share nearly identical
amino acid sequence, the inhibition potency of TGN-1 peptide is
almost 50 times higher than the Odriozola peptide (Cp23) by
comparing in vitro IC.sub.50 values (IC.sub.50 value for TGN-1 is
19.93 .mu.M and for Cp23 is .about.1033 .mu.M). Moreover, there was
nearly no difference in the IC.sub.50 values between the Odriozola
peptide (Cp23, 1033 .mu.M) and its scrambled peptide (Cp23-Der, 945
.mu.M). However, TGN-1 peptide showed much higher inhibitory effect
than its scrambled peptide TGN-4 (FIG. 2B), indicating that the
TGN-1 peptide shows sequence-specific inhibitory effect on in vitro
PTEN activity when the Odriozola peptide (Cp23) failed to do.
Additionally, TGN-2 peptide is different from the Odriozola peptide
(Cp23) by containing additional 15 amino acid residues including
the PDZ domain-binding motif, which is already shown to be
effective for PTEN inhibition (IC.sub.50 value for TGN-2 is 4.93
.mu.M). Also, TGN-1 and TGN-2 peptides include PTD (peptide
transfer domain) sequence at their N-terminal ends so that these
peptides can be introduced directly into the cells, whereas the
Odriozola peptides need to be fused with GFP and transfected into
the cells. Thus, TGN-1 and TGN-2 peptides possess effective PTEN
inhibition ability in vitro and in vivo.
[0037] We developed peptides by mimicking PTEN C-terminal region
including the "phosphorylation site". TGN-1 and TGN-2 showed
specific and effective inhibitory effect on PTEN activity in vitro
and up-regulated PI3K-Akt signaling pathway by blocking PTEN
activity in neuronal cells. Since facilitating PI3K-Akt-mTOR
signaling by suppression of PTEN is known to be effective in nerve
regeneration upon CNS injury [Saijilafu et al 2013], the inventive
peptides are useful as therapeutic or treatment agent for CNS
injury. Neurite assay using differentiated neuronal cells with TGN
peptides demonstrated that TGN-1 and TGN-2 peptides clearly show
neurotrophic effect, as well as neuroprotective effect on
degenerated neurite by enhancing neurite microtubule structure.
Thus, these peptides are therapeutic targets for nerve regeneration
after nerve injury including CNS injury, as well as for delaying
neurodegenerative progress.
[0038] Peptide Design
[0039] The inventive peptides, also referred to herein as "TGN
peptides", as PTEN inhibitor were designed using PTEN C-terminal
region (amino acid residues 352.about.403) as template.
[0040] It is preferred that all of the TGN peptides include PTD
(peptide transfer domain) sequence, which may include RRRRRRRR (SEQ
ID NO:2) at the N-terminal end to increase membrane
permeability.
[0041] The TGN peptide may be any fragment of PTEN within amino
acid residues 352.about.403 of PTEN amino acid sequence of SEQ ID
NO:1, or a fragment of PTEN that includes as part of its sequence,
a portion of the amino acid residues 352.about.403 of PTEN amino
acid sequence of SEQ ID NO:1. Preferably, the TGN peptide includes
phosphorylation of a Serine or Threonine present in this peptide
fragment. Preferably, the Serine or Threonine sites are at 366,
370, 380, 382, 383, or 385 of the PTEN protein of SEQ ID NO:1.
[0042] The TGN peptide may be at least 10 amino acid residues long,
at least 15, at least 20 at least 25, at least 30, at least 35, or
at least 40 amino acid residues long. It is preferred that
phosphorylation of at least one of the Serine or Threonine residue
or a combination thereof is included in the peptide.
[0043] It should be recognized that in one aspect, the TGN peptide
is not limited by the length of its peptide. It is preferred that
at least part of the peptide resides within amino acid residues 352
to 403.
[0044] In this regard, exemplified TGN-1 peptide has 24 amino acids
with three phosphorylated Serine residues
VTPDVpSDNEPDHYRYpSDTTDpSDPE (SEQ ID NO:3), pS=phosphorylated
Serine). When the PTD is attached at the N-terminus,
RRRRRRRR-VTPDVpSDNEPDHYRYpSDTTDpSDPE-amide (SEQ ID NO:4) is seen
having 32 amino acid residues.
[0045] Another exemplified peptide is TGN-2 peptide, which has 28
amino acids with two phosphorylated Serine residues
HYRYpSDTTDpSDPENEPFDEDQHTQITKV (SEQ ID NO:5).
[0046] When the PTD is attached at the N-terminus,
RRRRRRRR-HYRYpSDTTDpSDPENEPFDEDQHTQITKV-amide (SEQ ID NO:6) is seen
having 36 amino acid residues.
[0047] TGN-3 peptide has the same amino acid sequence as TGN-2
peptide but no residue is modified and two Serine residues were
substituted to Valine HYRYVDTTDVDPENEPFDEDQHTQITKV (SEQ ID NO:7).
When the PTD is attached at the N-terminus,
RRRRRRRR-HYRYVDTTDVDPENEPFDEDQHTQITKV-amide (SEQ ID NO:8) is
seen.
[0048] TGN-4 peptide was designed as a scrambled peptide of TGN-1
peptide SDDEYTDNPDSRYVSDTPVDTEH (SEQ ID NO:9). When the PTD is
attached at the N-terminus, RRRRRRRR-SDDEYTDNPDSRYVSDTPVDTEH-amide
(SEQ ID NO:10) is seen.
[0049] And TGN-5 peptide was designed for TGN-2/TGN-3 scrambled
peptide DEHDTEYTPDYRQETHFNSQPTDKSDVI (SEQ ID NO:11). When the PTD
is attached at the N-terminus,
RRRRRRRR-DEHDTEYTPDYRQETHFNSQPTDKSDVI-amide (SEQ ID NO:12) is
seen.
[0050] Chemically Modified Peptides
[0051] Polypeptide therapeutics may suffer from short circulating
half-life, and proteolytic degradation and low solubility. To
improve the pharmacokinetics and pharmacodynamics properties of the
inventive biopharmaceuticals, methods such as manipulation of the
amino acid sequence may be made to decrease or increase
immunogenicity and decrease proteolytic cleavage; fusion or
conjugation of the peptides to immunoglobulins and serum proteins,
such as albumin may be made; incorporation into drug delivery
vehicles for the biopharmaceuticals such as the inventive peptides
and antibodies for protection and slow release may also be made;
and conjugating to natural or synthetic polymers are also
contemplated. In particular, for synthetic polymer conjugation,
pegylation or acylation, such as N-acylation, S-acylation,
amidation and so forth are also contemplated.
[0052] Nerve Tissue
[0053] Nervous tissue derives from the embryonic ectoderm under the
influence of the notochord. The ectoderm is induced to form a
thickened neural plate that then differentiates and the ends
eventually fuse to form the neural tube from which all of the
central nervous system derives. The central nervous system consists
of the brain, cranial nerves and spinal cord. The peripheral
nervous system derives from cells next to the neural groove called
the neural crest.
[0054] Nerve tissue is distributed throughout the body in a complex
integrated communications network. Nerve cells (neurons)
communicate with other neurons in circuits ranging form very simple
to very complex higher-order circuits. Neurons do the actual
message transmission and integration while other nervous tissue
cells called glial cells assist neurons by support, protection,
defense and nutrition of the neurons. There are about 10 times more
glial cells than neurons in the brain. Glial cells create the
microenvironment needed for neuronal function and sometimes they
assist in neural processing and activity. Neurons are excitable
cells. This means that when properly stimulated, an action
potential can be initiated that may be propagated over the cell
membrane to transmit information to distant cells. Neurons are
independent functional units responsible for the reception,
transmission and processing of stimuli.
[0055] In general, neurons consist of three parts; the cell body,
where the nucleus and cellular organelles are located; dendrites,
which are processes extending from the cell body that receive
stimuli from the environment or other neurons; and the axon, which
is a long single process extending from the cell body for the
transmission of nerve impulses to other cells. The axon usually
branches at its distal end and each branch terminating on another
cell has a bulbous end. The interaction of the end bulb with the
adjacent cell forms a structure called a synapse. Synapses are
specialized to receive a signal and convert it into an electrical
potential.
[0056] Most neurons found in the human body are multipolar, meaning
they have more than two cell processes with only one being an axon
and the remaining processes being dendrites. Bipolar neurons of the
retina or olfactory mucosa have one dendritic process and an axon
coming off the cell body. Pseudounipolar neurons found in the
spinal cord ganglia enable sensory impulses picked up by the
dendrites to travel directly to the axon without passing through
the cell body. Neurons may also be classified according to
function. Sensory neurons are involved in the reception and
transmission of sensory stimuli. Motor neurons send impulses to
control muscles and glands. Other neurons, interneurons, act as
go-betweens between neurons as part of functional networks.
[0057] Synapses are specialized functional cell junctions to
propagate cellular signals. Most synapses are chemical synapses
where vesicles in the presynaptic terminal contain a chemical
messenger that is released to the synaptic cleft when the
presynaptic membrane is stimulated. The chemical messenger diffuses
across the synaptic cleft to bind to receptors in the postsynaptic
membrane. This induces a change in the polarization state of the
postsynaptic membrane effecting cellular action. A special type of
synapse is the neuromuscular junction. More than 35
neurotransmitters are known and most are small molecules (nitric
oxide, acetylcholine), catecholamines (norepinephrine, serotonin),
or neuroactive peptides (endorphin, vasopressin). Once used, the
neurotransmitters are removed quickly by enzymatic breakdown,
diffusion or endocytosis by the presynaptic cell.
[0058] Some neurons are wrapped in an insulating material called
myelin. This lipid rich material is formed by glial cells: Schwann
cells in the peripheral nervous system and by oligodendrocytes in
the central nervous system. The insulation enables faster nerve
conduction by reducing the membrane surface area that must be
depolarized. In myelinated neurons the nerve impulse jumps from one
unmyelinated segment to another over the length of the axon. It is
the myelin sheath and lack of neuron cell bodies within the tissue
that makes some nervous tissue appear white as in the large
peripheral nerves and white matter of the brain. Other glial cells,
called astrocytes, are involved in structural integrity, neuronal
nutrition and maintaining the microenvironment of nervous tissue.
Astrocytes, are in direct communication with one another via gap
junctions and can affect the survival of neurons in their care by
the regulation of the the local environment. Ependymal cells line
spinal cord and the ventricles of the brain and secrete the
cerebrospinal fluid. Other small glial cells, called microglia, are
phagocytic cells that are involved with inflammation and repair in
the adult central nervous system.
[0059] Nervous tissue is an excitable tissue that is capable of
receiving and transmitting electrical impulses. The central cell
type is called a neuron. Neurons usually have a cell body,
dendrites that receive inputs, and an axon that transmits
electrical potentials.
[0060] Neurons may be classified as sensory, motor, secretory or
association neurons. They are often classified by conduction speed,
diameter and the presence or absence of specialized lipoprotein
insulation called myelin. Type A fibers are myelinated and can
conduct impulses at 12-120 m/sec. Type B are also myelinated fibers
but they only transmit impulses at 3-5 m/sec. Type C fibers are
unmyelinated, small in diameter and very slow (2.5 m/sec). An
example of a Type A fiber is a motor neuron innervating the
gastrocnemius. An autonomic preganglionic efferent neuron is an
example of a Type B fiber and a sensory neuron carrying information
about diffuse pain is an example of a slow Type C fiber.
[0061] Sensory neurons are adapted to detect certain types of
information from the environment. These include mechanoreceptors
sensing things like pressure or stretch, thermoreceptors,
photoreceptors in the retina, and chemoreceptors such as the taste
bud or those for olfaction. Association neurons, or interneurons
are usually found in the spinal cord and brain where they connect
sensory afferent neurons to efferent motor or secretory
neurons.
[0062] Neurons communicate with one another via a structure called
the synapse. An axon ends in one or more terminal buttons that
contain numerous small vesicles. These small vesicles are filled
with chemical substances called neurotransmitters. Acetylcholine is
most often the neurotransmitter at the synapse although other
chemicals like norepinephrine, serotonin and GABA may be used
dependent on the neuron. When an impulse travels down the axon and
reaches the terminal buttons the vesicles fuse with the neuronal
membrane and the neurotransmitter is released. The chemical then
diffuses across the narrow synaptic cleft to specific receptors for
the chemical on the postsynaptic membrane of the receiving
neuron.
[0063] The interaction of the neurotransmitter with the receptor
causes a change in the membrane potential that may induce a new
impulse postsynaptic neuron. The enzyme acetylcholinesterase is
present in synapse to break down acetycholine and terminate the
stimulus. Other neurotransmitters are either broken down or taken
back up into the presynaptic neuron to terminate the stimulus.
[0064] In the central nervous system many neurons may converge on a
single neuron. When each of the presynaptic neurons releases
neurotransmitter into its synapse with the postsynaptic neuron,
local membrane potentials occur that are integrated and summed.
These incoming signals may be inhibitory or stimulatory. If the
resulting summed membrane potential reaches the minimum threshold
for that neuron, then an action potential will be initiated.
[0065] Action potentials travel in one direction away from the cell
body by saltatory conduction. The fastest neurons are covered in
myelin sheaths arranged in discreet segments separated by nodes of
naked neuronal membrane called nodes of Ranvier. In saltatory
conduction, the electrical potential jumps from node to node,
thereby reducing the membrane area involved in conduction of the
action potential and speeding up conduction.
[0066] Non-neural cells found in the nervous system are called
glial cells. Astrocytes are the most numerous and provide support
and nourishment of neurons. Microglia are small phagocytic cells
specific to neural tissue. Cells that line the ventricular system
and central canal of the spinal cord and make cerebrospinal fluid
are called ependymal cells. In the central nervous system, an
oligodendrocyte forms segments of the myelin sheaths of multiple
neurons. In the peripheral nervous system, each segment of the
myelin sheath is made by a single Schwann cell.
[0067] Central Nervous System
[0068] The central nervous system (CNS) consists of the brain and
spinal cord. The meninges (dura mater, arachnoid and pia mater)
protect and nourish the CNS in addition to the protection afforded
by the bony skull and vertebrae. Cerebrospinal fluid is found in
the the subarachnoid space, central canal of the spinal column and
the ventricles of the brain. The pia mater is the innermost layer
and is adherant to the nervous tissue. Between the pia mater and
the dura mater lies the arachnoid layer. The tough fibrous dura
mater lies just beneath the skull.
[0069] The brain can be divided into 3 basic areas of the
forebrain, midbrain, and brain stem. The forebrain includes the
thalamus, hypothalamus, basal ganglia, and cerebrum. The cerebrum
is responsible for conscious thought, interpretation of sensations,
all voluntary movements, mental faculties, and the emotions.
[0070] Cerebral tissue can be divided into structural and
functional areas. The surface of the cerebrum is convoluted into
gyri (ridges) and sulci (grooves). The cortical sensory and motor
areas can be mapped to the post central gyms and central sulcus,
respectively. The sensory area receives sensory info from the
opposite side of the body that is projected after thalamic
processing. Those parts of the body with more sensory nerve endings
are represented by more cortical sensory area. The motor area
controls voluntary muscle movements of the contralateral body parts
but the association areas are important for the initiation of
movement.
[0071] The cerebrum is the largest part of the brain and is divided
into two hemispheres, right and left, having several lobes. The
frontal lobe contains the motor area, Broca's speech area,
association areas, and functions in intelligence and behavior. The
parietal lobe contains sensory areas and function in feeling and
hearing. Primary visual association areas are located in the
occipital lobe and the temporal lobe contains areas for auditory
association, smell and memory storage.
[0072] The thalamus is located between the cerebral cortex and
brainstem. All sensory input except the sense of smell is processed
here before being projected to other areas of the brain. The
hypothalamus is located beneath the thalamus and is responsible for
processing internal stimuli and the maintenance of the internal
environment. Moment by moment unconscious control of blood
pressure, temperature, heart rate, respiration, water metabolism,
osmolality, hunger, and neuroendocrine activities are handled here.
Nuclei of the neuroendocrine cells that release oxytocin and ADH
from the posterior pituitary are located in the hypothalamus.
[0073] The basal ganglia (caudate nucleus, globus palladus,
substantia nigra, subthalamic nucleus, red nucleus) are groups of
neurons embedded within each hemisphere of the cerebrum. They are
involved in the control of complex motor control, information
processing and unconscious gross intentional movements.
[0074] The brainstem includes the medulla oblongata and pons. The
medulla oblongata contains important functional areas and relay
centers for the control of respiration, cardiac and vasomotor
reflexes. The pons contains the pneumotaxic center which is
involved in the regulation of respiration.
[0075] The cerebellum lies above the brainstem and uses sensory
information processed elsewhere about the position of the body,
movement, posture and equilibrium. Movements are not initiated in
the cerebellum but it is necessary for coordinated movement.
[0076] Peripheral Nervous System
[0077] The peripheral nervous system includes nerves, ganglia,
spinal and cranial nerves located outside the brain and spinal
cord. The twelve cranial nerves arise from nuclei located in the
brainstem and travel to specific locations carrying impulses to
control various autonomic functions like smell, vision, salivation,
heart rate and cutaneous sensation. Cranial nerves are often mixed
in that they carry sensory and motor components but they may have
only motor or sensory fibers. The following table lists the cranial
nerves and their functions.
TABLE-US-00002 TABLE 1 Cranial Nerves Number Name Function I
Olfactory Sense of smell II Optic Vision III Oculomotor Motor
control of some eye muscles and eyelid IV Trochlear Motor control
of some eye muscles V Trigeminal Chewing muscles and some facial
sensation VI Abducent Motor control of some eye muscles VII Facial
Motor control of facial muscles, salivation. Taste and cutaneous
sensations. VIII Acoustic Equilibration, static sense and hearing
IX Glossopharyngeal Salivation, sensations of skin, taste and
viscera X Vagus Motor control of the heart and viscera, sensation
from the thorax, pharynx and abdominal viscera XI Accessory Motor
impulses to the pharynx and shoulder XII Hypoglossal Motor control
of the tongue, some skeletal muscles, some viscera, sensation from
skin and viscera
[0078] The sensory division of the peripheral nervous system takes
input from various types of receptors, processes it and sends to
the central nervous system. Sensory input can come from internal
sources as in proprioception (sense of position of the joints and
muscles) or external sources as in the sensation of pressure or
heat on the skin. Areas of the skin innervated by specific spinal
nerves are called dermatomes. Afferent fibers collect sensory input
and travel up the spinal cord, converge in the thalamus, and end
finally on the sensory cortex of the cerebrum. Those areas with
more sensory receptors, i.e. the fingertips or lips, correspond to
a larger area on the sensory cortex of the brain. Fibers carrying
proprioceptive information are dispersed to the cerebellum as well.
Almost all sensory systems transmit impulses to parts of the
thalamus. The cerebral cortex is involved in conscious perception
and interpretation of sensory stimuli.
[0079] Motor inputs to muscles and glands occur via the autonomic
and somatic efferent systems. CNS innervation of the joints,
tendons and muscles travel via the somatic efferent system. Some
muscular responses are handled via spinal reflexes. An example of
this is the withdrawal reflex seen when the finger contacts a hot
stove. The movement to remove the finger occurs via a simple spinal
reflex long before the sensation of pain reaches the brain. Clearly
this is protective mechanism to avoid further injury. Motor inputs
to glands and smooth muscle usually occur via the autonomic
system.
[0080] Most organs receive input from both branches of the
autonomic nervous system. One branch will generally be excitatory
while the other is inhibitory in that organ or tissue. The
sympathetic branch of the autonomic system acts to prepare the body
for physiologic stress. Stimulation of the sympathetic branch is
like stepping on the gas in that the body prepares to run or fight
in response. Effects such as an increased heart rate, dilation of
airways and mobilization of glucose from glycogen stores are seen.
Sympathetic nerves arise from the 1.sup.st thoracic to the 4.sup.th
lumbar vertebra. They have a short preganglionic neuron that ends
in one of the chain ganglia that lie along the spinal column.
Acetylcholine is the neurotransmitter at the synapse with the long
postganglionic neuron which then travels to the target tissue where
norepinephrine is released at the majority of sympathetic nerve
endings. A few sympathetic post ganglionic neurons, such as those
innervating sweat glands or skeletal muscle vasculature, release
acetylcholine.
[0081] The parasympathetic branch acts to counterbalance the
sympathetic branch via neurons that arise from the cranial and
sacral regions of the CNS. For instance, parasympathetic
stimulation constricts airways and decreases heart rate. It
regulates resting activities such as digestion, micturation and
erection. Long preganglionic neurons release acetylcholine at
synapses close to the end organ. Short postganglionic neurons also
release acetylcholine on the effector tissue.
[0082] Therapeutic Composition
[0083] In one embodiment, the present invention relates to
treatment for various diseases that are characterized by
neurodegeneracy. In this way, the inventive therapeutic compound
may be administered to human patients who are either suffering
from, or prone to suffer from the disease by providing compounds
that inhibit neuronal degeneration. In particular, the disease is
associated with neurodegenerative disorder of the brain, loss of
nerve cell, particularly in the hippocampus and cerebral cortex,
reduced neurotransmitters, cerebrovascular degeneration, crushed
nerve in the spine, and/or loss of cognitive ability.
[0084] The formulation of therapeutic compounds is generally known
in the art and reference can conveniently be made to Remington's
Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton,
Pa., USA. For example, from about 0.05 .mu.g to about 20 mg per
kilogram of body weight per day may be administered. Dosage regime
may be adjusted to provide the optimum therapeutic response. For
example, several divided doses may be administered daily or the
dose may be proportionally reduced as indicated by the exigencies
of the therapeutic situation. The active compound may be
administered in a convenient manner such as by the oral,
intravenous (where water soluble), intramuscular, subcutaneous,
intra nasal, intradermal or suppository routes or implanting (eg
using slow release molecules by the intraperitoneal route or by
using cells e.g. monocytes or dendrite cells sensitised in vitro
and adoptively transferred to the recipient). Depending on the
route of administration, the peptide may be required to be coated
in a material to protect it from the action of enzymes, acids and
other natural conditions which may inactivate said ingredients.
[0085] For example, the low lipophilicity of the peptides will
allow them to be destroyed in the gastrointestinal tract by enzymes
capable of cleaving peptide bonds and in the stomach by acid
hydrolysis. In order to administer peptides by other than
parenteral administration, they will be coated by, or administered
with, a material to prevent its inactivation. For example, peptides
may be administered in an adjuvant, co-administered with enzyme
inhibitors or in liposomes. Adjuvants contemplated herein include
resorcinols, non-ionic surfactants such as polyoxyethylene oleyl
ether and n-hexadecyl polyethylene ether. Enzyme inhibitors include
pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) and
trasylol. Liposomes include water-in-oil-in-water CGF emulsions as
well as conventional liposomes.
[0086] The active compounds may also be administered parenterally
or intraperitoneally. Dispersions can also be prepared in glycerol
liquid polyethylene glycols, and mixtures thereof and in oils.
Under ordinary conditions of storage and use, these preparations
contain a preservative to prevent the growth of microorganisms.
[0087] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions (where water soluble) or dispersions and
sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersion. In all cases the form must be
sterile and must be fluid to the extent that easy syringability
exists. It must be stable under the conditions of manufacture and
storage and must be preserved against the contaminating action of
microorganisms such as bacteria and fungi. The carrier can be a
solvent or dispersion medium containing, for example, water,
ethanol, polyol (for example, glycerol, propylene glycol and liquid
polyethylene glycol, and the like), suitable mixtures thereof, and
vegetable oils. The proper fluidity can be maintained, for example,
by the use of a coating such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by the use of
superfactants. The prevention of the action of microorganisms can
be brought about by various antibacterial and antifungal agents,
for example, chlorobutanol, phenol, sorbic acid, theomersal and the
like. In many cases, it will be preferable to include isotonic
agents, for example, sugars or sodium chloride. Prolonged
absorption of the injectable compositions can be brought about by
the use in the composition of agents delaying absorption, for
example, aluminium monostearate and gelatin.
[0088] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterile
active ingredient into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum drying and the freeze-drying technique
which yield a powder of the active ingredient plus any additional
desired ingredient from a previously sterile-filtered solution
thereof.
[0089] When the peptides are suitably protected as described above,
the active compound may be orally administered, for example, with
an inert diluent or with an assimilable edible carrier, or it may
be enclosed in hard or soft shell gelatin capsule, or it may be
compressed into tablets, or it may be incorporated directly with
the food of the diet. For oral therapeutic administration, the
active compound may be incorporated with excipients and used in the
form of ingestible tablets, buccal tablets, troches, capsules,
elixirs, suspensions, syrups, wafers, and the like. Such
compositions and preparations should contain at least 1% by weight
of active compound. The percentage of the compositions and
preparations may, of course, be varied and may conveniently be
between about 5 to about 80% of the weight of the unit. The amount
of active compound in such therapeutically useful compositions is
such that a suitable dosage will be obtained. Preferred
compositions or preparations according to the present invention are
prepared so that an oral dosage unit form contains between about
0.1 .mu.g and 2000 mg of active compound.
[0090] The tablets, pills, capsules and the like may also contain
the following: A binder such as gum tragacanth, acacia, corn starch
or gelatin; excipients such as dicalcium phosphate; a
disintegrating agent such as corn starch, potato starch, alginic
acid and the like; a lubricant such as magnesium stearate; and a
sweetening agent such as sucrose, lactose or saccharin may be added
or a flavoring agent such as peppermint, oil of wintergreen, or
cherry flavoring. When the dosage unit form is a capsule, it may
contain, in addition to materials of the above type, a liquid
carrier. Various other materials may be present as coatings or to
otherwise modify the physical form of the dosage unit. For
instance, tablets, pills, or capsules may be coated with shellac,
sugar or both. A syrup or elixir may contain the active compound,
sucrose as a sweetening agent, methyl and propylparabens as
preservatives, a dye and flavoring such as cherry or orange flavor.
Of course, any material used in preparing any dosage unit form
should be pharmaceutically pure and substantially non-toxic in the
amounts employed. In addition, the active compound may be
incorporated into sustained-release preparations and
formulations.
[0091] As used herein "pharmaceutically acceptable carrier and/or
diluent" includes any and all solvents, dispersion media, coatings
antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like. The use of such media and agents for
pharmaceutical active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active ingredient, use thereof in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions.
[0092] It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
mammalian subjects to be treated; each unit containing a
predetermined quantity of active material calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on (a) the
unique characteristics of the active material and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an active material for the treatment
of disease in living subjects having a diseased condition in which
bodily health is impaired.
[0093] The principal active ingredient is compounded for convenient
and effective administration in effective amounts with a suitable
pharmaceutically acceptable carrier in dosage unit form. A unit
dosage form can, for example, contain the principal active compound
in amounts ranging from 0.5 .mu.g to about 2000 mg. Expressed in
proportions, the active compound is generally present in from about
0.5 .mu.g/ml of carrier. In the case of compositions containing
supplementary active ingredients, the dosages are determined by
reference to the usual dose and manner of administration of the
said ingredients.
[0094] Delivery Systems
[0095] Various delivery systems are known and can be used to
administer a compound of the invention, e.g., encapsulation in
liposomes, microparticles, microcapsules, recombinant cells capable
of expressing the compound, receptor-mediated endocytosis,
construction of a nucleic acid as part of a retroviral or other
vector, etc. Methods of introduction include but are not limited to
intradermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, intranasal, epidural, and oral routes. The compounds
or compositions may be administered by any convenient route, for
example by infusion or bolus injection, by absorption through
epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and
intestinal mucosa, etc.) and may be administered together with
other biologically active agents. Administration can be systemic or
local. In addition, it may be desirable to introduce the
pharmaceutical compounds or compositions of the invention into the
central nervous system by any suitable route, including
intraventricular and intrathecal injection; intraventricular
injection may be facilitated by an intraventricular catheter, for
example, attached to a reservoir, such as an Ommaya reservoir.
Pulmonary administration can also be employed, e.g., by use of an
inhaler or nebulizer, and formulation with an aerosolizing
agent.
[0096] In a specific embodiment, it may be desirable to administer
the pharmaceutical compounds or compositions of the invention
locally to the area in need of treatment; this may be achieved by,
for example, and not by way of limitation, local infusion during
surgery, topical application, e.g., in conjunction with a wound
dressing after surgery, by injection, by means of a catheter, by
means of a suppository, or by means of an implant, said implant
being of a porous, non-porous, or gelatinous material, including
membranes, such as sialastic membranes, or fibers. Preferably, when
administering a protein, including an antibody or a peptide of the
invention, care must be taken to use materials to which the protein
does not absorb. In another embodiment, the compound or composition
can be delivered in a vesicle, in particular a liposome. In yet
another embodiment, the compound or composition can be delivered in
a controlled release system. In one embodiment, a pump may be used.
In another embodiment, polymeric materials can be used. In yet
another embodiment, a controlled release system can be placed in
proximity of the therapeutic target, i.e., the brain, thus
requiring only a fraction of the systemic dose.
[0097] A composition is said to be "pharmacologically or
physiologically acceptable" if its administration can be tolerated
by a recipient animal and is otherwise suitable for administration
to that animal. Such an agent is said to be administered in a
"therapeutically effective amount" if the amount administered is
physiologically significant. An agent is physiologically
significant if its presence results in a detectable change in the
physiology of a recipient patient.
[0098] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and accompanying figures. Such modifications
are intended to fall within the scope of the appended claims. The
following examples are offered by way of illustration of the
present invention, and not by way of limitation.
EXAMPLES
Example 1--Materials and Experimental Methods
Example 1.1
[0099] Rat adrenal medullary PC12 pheochromocytoma neuronal cell
was purchased from ATCC (Manassas, Va.). Cell culture materials
including Dulbecco's modified Eagle's medium (DMEM), fetal bovine
serum (FBS) and horse serum were purchased from Mediatech Inc.
(Manassas, Va.). 2.5 S Nerve growth factor was purchased from BD
Biosciences, Inc. (Bedford, Mass. 01730). TUJ-1 monoclonal rabbit
antibody against neuronal class III -tubulin was purchased from
Covance Inc. (Gaithersburg, Md.). Monoclonal mouse antibody against
acetylated .alpha.-Tubulin was purchased from Santa Cruz Biotech
Inc. (Santa Cruz, Calif.). Goat serum, Texas Red.RTM. Goat
Anti-Rabbit IgG antibody, Alexa Fluor.RTM. 488 Goat anti-Mouse IgG
antibody, 4',6-Diamidino-2-Phenylindole, Dilactate (DAPI) and
AlamarBlue.RTM. were purchased from Molecular Probes-Invitrogen
(Eugene, Oreg.). Nocodazole was purchased from Sigma-Aldrich (St.
Louis, Mo.). Neurite Outgrowth Assay Kit was purchased from
Millipore (Billerica, Mass.). All lipids were purchased from Avanti
Polar Lipids, Inc. (Alabaster, Ala. 35007). Recombinant human PTEN
protein and Malachite Green phosphate detection kit were purchased
from R&D Systems, Inc. (Minneapolis, Minn. 55413). Human PTEN
c-DNA was purchased from OriGene Inc. (Rockville, Md. 20850).
Lipofectamine.TM. 2000 Transfection Reagent was purchased from
Invitrogen.TM.. Tris-Glycine gradient mini gel (.about.20%) was
purchased from Novex.TM.. All antibodies were purchased from Santa
Cruz Biotechology, Inc. (Santa Cruz, Calif. 95060). All other
materials were purchased from Fisher Scientific Inc.
Example 1.2--Peptide Design
[0100] TGN peptides as potential PTEN inhibitor were designed using
PTEN C-terminal region (AA352.about.403) as template. All TGN
peptides include PTD (peptide transfer domain) sequence (RRRRRRRR)
at their N-terminal end to increase membrane permeability. TGN-1
peptide has 32 amino acids with three phosphorylated Serine
residues (MW=4244.18 Da, sequence:
RRRRRRRR-VTPDVpSDNEPDHYRYpSDTTDpSDPE-amide (SEQ ID NO:4),
pS=phosphorylated Serine). TGN-2 peptide has 36 amino acids with
two phosphorylated Serine residues (MW=4776.28 Da, sequence:
HYRYpSDTTDpSDPENEPFDEDQHTQITKV-amide (SEQ ID NO:6),
pS=phosphorylated Serine). TGN-3 peptide has the same amino acid
sequence as TGN-2 peptide but no residue is modified and two Serine
residues were substituted to Valine (MW=4640.99 Da, sequence:
RRRRRRRR-HYRYVDTTDVDPENEPFDEDQHTQITKV-amide (SEQ ID NO:8)). TGN-4
peptide was designed as a scrambled peptide of TGN-1 peptide
(MW=4004.19 Da, sequence=RRRRRRRR-SDDEYTDNPDSRYVSDTPVDTEH-amide
(SEQ ID NO:10)) and TGN-5 peptides was designed for TGN-2/TGN-3
scrambled peptide (MW=4616.88 Da,
sequence=RRRRRRRR-DEHDTEYTPDYRQETHFNSQPTDKSDVI-amide (SEQ ID
NO:12)). All peptides were synthesized by 21.sup.st Century
Biochemicals Inc. (Marlboro, Mass. 01752). Purity was >95% and
confirmed by HPLC.
Example 1.3--In Vitro PTEN Activity Assay
[0101] In vitro PTEN activity assay was designed to check PTEN
lipid phosphatase activity to convert phosphatidylinositol
triphosphate (PIPS) to phosphatidylinositol diphosphate (PIP2) and
produce phosphate ion (Pt).
1,2-dioctanoyl-sn-glycero-3-phospho-(1'-myo-inositol-3,4,5-triphosp-
hate) (C8-PIP3) was used as PTEN substrate and prepared as lipid
vesicle (liposome) with other phospholipids because PTEN as lipid
phosphatase is an interfacial enzyme. For liposome preparation,
C8-PIP3, DOPS (1,2-dioeloyl-sn-glycero-phosphoserine) and DOPC
(1,2-dioeloyl-sn-glycero-phosphocholine) were mixed together with
800 .mu.L of liposome buffer (50 mM Tris, 100 mM NaCl, 10 mM
MgCl.sub.2, 5 mM DTT, pH=8.0) to final concentration of 0.1 mM of
C8-PIP3, 0.25 mM DOPS and 0.25 mM DOPC. The lipid mixture was then
sonicated at 4.degree. C. for 30 min to produce liposome. After
sonication, the liposome solution was briefly centrifuged to remove
remaining lipids.
[0102] For PTEN activity assay, 20 ng of recombinant human PTEN
protein was mixed with 40 .mu.L of completed liposome solution.
PTEN assay buffer (1 mM Tris, 20 mM DTT and 0.5% NP-40, pH=8.0) was
added up to 100 .mu.L as final volume. The reaction mixture then
was incubated at 37.degree. C. water bath for 30 min. After
incubation, the inorganic phosphate ions produced by PTEN protein
was detected using Malachite Green phosphate detection kit.
Firstly, 50 or 100 .mu.L of each reaction mixture was transferred
to 96-well plate and 10 or 20 .mu.L of Malachite reagent A,
respectively, was added and incubated at room temperature for 10
min. After the incubation was finished, 10 or 20 .mu.L of Malachite
reagent B was added again to each sample and further incubated for
20 minutes at room temperature. Detection of the phosphate ions was
performed by measuring OD (optical density) at 620 nm using
spectrophotometer. For determining the inhibitory effect of TGN
peptides (10 .mu.M) on recombinant PTEN activity, each TGN peptide
was prepared in DMSO solution at 1 mM concentration, and 1 .mu.L of
the TGN peptide solution was mixed with recombinant PTEN protein,
liposome and PTEN assay buffer and assayed for PTEN activity by
following the above protocol.
Example 1.4--In Vitro IC.sub.50 Assay
[0103] IC.sub.50 values were measured by performing in vitro PTEN
activity assay with different concentrations of TGN-1 and TGN-2
peptides. The concentration range of TGN-1 or TGN-2 peptides for
IC.sub.50 assay were 0.1, 1, 10, 30, 60, and 100 .mu.M and 0.05,
0.1, 0.5, 1, 5, 10, and 100 .mu.M, respectively. All data represent
experimentation in triplicate and the IC.sub.50 values were
calculated by Prism 5 software (GraphPad Software).
Example 1.5--PC 12 Cell Culturing
[0104] PC12 rat pheochromocytoma cells were seeded to 6-well plate
(0.6.times.10.sup.6 cells/well) and cultured with DMEM media
containing 7.5% FBS and 7.5% Goat Serum. After the cell confluency
reached around 60.about.70%, NGF (nerve growth factor, 50 ng/mL)
was added to the PC12 cells for differentiation and incubated for 5
more days. Then, fresh media containing different amounts of TGN
peptides in DMSO solution were added to each well and incubated
further for 24 hr. For PTEN overexpression, PC12 cells were seeded
in 6-well plate (1.0.times.10.sup.6 cells/well) and differentiated
with NGF (50 ng/mL) as above. DNA-Lipofectamine 2000 mixture was
prepared for each well of cells to be transfected by firstly adding
2.about.2.5 .mu.g of human PTEN c-DNA into 500 .mu.l of Opti-MEM.
3.75-8.75 .mu.l of Lipofectamine 2000.TM. reagent was added next to
the above diluted DNA solution, mixed gently and incubated for 25
minutes at room temperature. Growth media of PC12 cells in 6-well
plate was exchanged with fresh media and 500 .mu.l of the
DNA-Lipofectamine 2000 complex was added to each well for
transfection. Transfected cells were incubated at 37.degree. C. in
5.0% CO.sub.2 incubator for 24-48 hours post-transfection before
assaying for transgene expression.
Example 1.6--Neurite Assay with PC12 Cells
[0105] Rat adrenal medullary PC12 rat pheochromocytoma neuronal
cells were supplemented with 7.5% fetal bovine serum (FBS), 7.5%
horse serum (ES) and 0.5% penicillin streptomycin in T-75 cm.sup.2
flasks that were maintained at 37.degree. C. in a 5% CO.sub.2
incubator. Cells were split at 50% confluence by gently
mechanically detaching them from the flask and propagated at a
split ratio 1:7.
[0106] For neurite protection assay, PC12 cells were seeded to
6-well plates with seeding density of 2.08.times.10.sup.5
cells/scaffold (empirically determined as optimal seeding density)
and incubated for 24-48 hr until cell confluency was reached to
60.about.70%. PC12 cells were then differentiated with NGF (50
ng/mL) for 72-120 hr. To mimic neurite degeneration, the
differentiated PC12 cells were treated with Nocodazole (0.5 .mu.M).
After 1 hr incubation at 37.degree. C., the old media containing
Nocodazole were switched with fresh media containing NGF (long/mL)
and/or TGN peptides (100 .mu.M as final concentration) and for
additional 72 hrs. Remaining neurites were analyzed via
immunofluorescence assay described below.
[0107] For neurite outgrowth assay, PC12 cells were seeded to
6-well plate with 1.0.times.10.sup.5 cells/well seeding density.
After cell confluence reached 60.about.70%, differentiation of the
PC12 cells was initiated by adding NGF (50 ng/mL). After 24 hr of
incubation, TGN peptides (50 .mu.M as final concentration) were
added to the wells in 6-well plates and incubated for two
additional days. Neurite status was quantified with
spectrophotometer using Neurite Outgrowth Kit (Millipore) described
below.
Example 1.7--Western Blotting
[0108] After culturing, PC12 cells were collected from the 6-well
plate and centrifuged down with bench-top centrifuger to make cell
pellet (13,000 rpm, 5 min at RT). Supernatant was discarded and the
cell pellet was resuspended with 3.about.500 .mu.L of 1.times.PIPA
buffer (Invitrogen). Resuspended cells were lysed by freezing-thaw
cycle using liquid nitrogen and 37.degree. C. water bath (3-4
times), followed by repeated spraying of resuspended cells using
syringe with 27G needle. The lysed cells were centrifuged at 10,000
g for 20 min at 4.degree. C. and the supernatants were collected
and assayed for total protein concentration using BCA protein
concentration kit (Thermo Scientific.).
[0109] Western blotting was performed to examine the
phosphorylation level of endogenous Akt protein in PC12 cells using
anti-phospho Akt antibody. SDS-PAGE was performed using Novex.TM.
gradient mini gel (10.about.20%). The cell lysate samples and
proteins in SDS-PAGE gel were transferred on to PVDF membrane,
followed by incubation with blocking solution (5% milk in
1.times.TBS buffer containing 0.1% Tween-20). Anti-phospho Akt
antibody was used as primary antibody with 1:500 dilution
(1.times.TBS buffer containing 0.1% Tween-20). HRP-conjugated
anti-rabbit antibody was used as secondary antibody with 1:8000
dilution factor. The expression level of endogenous or
overexpressed PTEN protein was also examined using anti-PTEN
antibody (1:400 dilution factor). .beta.-actin expression level was
also assayed for loading control.
Example 1.8--Neurite Quantification
[0110] For quantification of total neurites, we used Neurite
Outgrowth Assay Kit (Millipore) with spectrophotometer. After the
underside of the Millicell inserts (EMD Millipore, Billerica,
Mass., USA) was coated with fresh extracellular matrix (ECM)
protein (10 .mu.g/mL collagen) for 2 hours at 37.degree. C., PC12
cells were seeded per insert, that were placed into each well of a
24 well plate. Cells were kept at room temperature for 15 minutes
for attachment, and then a total of 700 .mu.l differentiation
medium was added per well (600 .mu.l and 100 .mu.l, below and above
the membrane, respectively). Neurites were left to extend for 3
days and then the inserts were fixed with -200.degree. C. methanol
for 20 minutes at room temperature, followed by fresh PBS rinse.
Next, inserts were placed into 400 .mu.l neurite staining solution
for 30 minutes at room temperature, and after cell bodies were
removed by a moistened cotton swab, each insert was placed onto 100
.mu.l Neurite Stain Extraction Buffer (Millipore). Finally, the
solutions were transferred into a 96 well plate and quantified on a
spectrophotometer by reading absorbance at 562 nm.
Example 1.9--Immunofluorescence
[0111] After cell culture, growth media were removed and the cells
were fixed with 10% formalin at room temperature for 15 minutes.
Afterward, the cells were washed with a 0.5M glycine solution in
PBS and blocked overnight at 40.degree. C. with 5% Goat Serum and
0.2% Triton-X solution in PBS. For immunostaining with primary
antibodies, cells were incubated overnight at 40.degree. C. with
TUJ-1 monoclonal rabbit antibody against neuronal class III
-tubulin (1:200 dilution) for total neurite staining and with
monoclonal mouse antibody against acetylated .alpha. Tubulin (1:100
dilution) for stable neurite staining. Once cells were washed three
times with 1.times.PBS buffer (10 minutes/wash), secondary
antibodies--Texas Red.RTM. goat anti rabbit IgG (1:200 dilution)
for TUJ-1 antibody and Alexa Fluor.RTM. 488 goat anti mouse IgG
(1:200 dilution) for acetylated .alpha. Tubulin antibody--were
added and incubated overnight at 40.degree. C. Subsequently, the
cells were washed three times in 1.times.PBS buffer (10
minutes/wash) and 1 .mu.g/ml 4',6-Diamidino-2-Phenylindole;
Dilactate (DAPI) was added after the second washing step for
staining cell nuclei. After final washing, cells were prepared to
be examined using fluorescence microscope. The excitation and
emission wavelengths are 488 nm/519 nm for Alexa Fluor.RTM. 488-IgG
(green), and 595/615 nm for Texas Red.RTM. goat anti rabbit IgG
(red) and 405/461 nm for DAPI. Fluorescence images of the cells
were acquired at different magnifications and analyzed by "ImageJ"
image processing and analysis program (Public Domain by Wayne
Rasband, NIH, Bethesda, Md., USA).
Example 2--Results
Example 2.1--TGN Peptides were Designed Using PTEN Phosphorylation
Site as Template
[0112] Blocking of PTEN activity as lipid phosphatase in vivo is
known to be effective in axon regeneration after nerve injury [Park
et. al 2008, Christie et. al 2012]. We investigated PTEN-membrane
association mechanism for designing potential PTEN inhibitor that
blocks PTEN localization on cell membrane surface. According to
previous studies [Lee et. al 1999; Leslie et. al 2008], PTEN
protein has two functional domains--phosphatase domain and C2
domain--and also possesses "phosphorylation site" in the C-terminal
region, which acts as a "switch" to control conformational change
of PTEN protein via phosphorylation-dephosphorylation process [Das
et. al 2003; Leslie et. al 2008]. For full lipid phosphatase
activity of PTEN, dephosphorylation of phosphorylated
serine/tyrosine residues at the "phosphorylation site should occur
in order to change PTEN conformation before PTEN-membrane
association. Additional binding via N-terminal PIP2 binding motif
and C-terminal PDZ domain binding motif localizes PTEN protein on
cell membrane in appropriate position required for full PTEN
activity [Walker et. al 2004; Molina et. al 2010]. Thus, we decided
to use PTEN "phosphorylation site" plus PDZ-domain binding motif as
a template for designing TGN peptides as potential PTEN inhibitor
by disrupting PTEN-membrane association (FIG. 1A).
[0113] TGN-1 peptide mimics the amino acid sequence (365-388) of
the "phosphorylation site" and TGN-2 and TGN-3 peptides mimic the
amino acid sequence (376-403) of C-terminal region including the
"phosphorylation site" and PDZ domain binding motif(399-403). Since
phosphorylation at serine residues in the "phosphorylation site" is
critical for PTEN conformation change [Leslie et. al 2008;
Odriozola et. al 2007], TGN-1 peptide is modified to include three
Serine residues phosphorylated (Ser 370, Ser380 and Ser385) inside
the "phosphorylation site". TGN-2 peptide includes two
phosphorylated serine residues (Ser380 and Ser385). In TGN-3
peptide, two serine residues (Ser380 and Ser385) were exchanged to
Valine for comparison. TGN-4 and TGN-5 peptide were designed to
scramble TGN-1 and TGN-2 peptide sequences, respectively. All TGN
peptides were also modified to be include eight Arginine residues
as peptide transfer domain (PTD) at the N-terminus to increase cell
membrane permeability (FIG. 1B).
Example 2.2--TGN-1 and TGN-2 Peptides Shows Specific Inhibitory
Effect on In Vitro PTEN Activity
[0114] Synthesized TGN peptides were tested for their PTEN
inhibitory effect using in vitro PTEN activity assay. Di-octanoyl
phosphatidylinositol 3,4,5 triphosphate (diC8-PIP.sub.3) was chosen
as a substrate for PTEN and prepared as lipid vesicle (liposome)
with two different phospholipids--dioleoyl phosphatidylcholine
(DOPC) and dioleoyl phosphatidylserine (DOPS). Lipids were mixed
with liposome buffer and became liposome by sonication (total lipid
concentration=0.6 mM). Prepared liposome (0.1 mM of di-C8 PIP3) was
incubated with 20 ng of recombinant human PTEN protein for 30
minutes at room temperature to assay for PTEN activity by
converting C8-PIP3 to C8-PIP2 and producing phosphate ions. The
phosphate ions produced by PTEN were measured using Malachite Green
reagent kit (FIG. 2A). 10 .mu.M of each TGN peptide was examined
for its inhibitory effect on PTEN activity. As seen in FIG. 2B,
both TGN-1 and TGN-2 peptides significantly blocked PTEN activity
(PTEN activity was decreased to 54% with TGN-1 and 31% with TGN-2
compared with positive control). On the other hand, TGN-2 peptide
showed limited inhibition compared with TGN-1 or TGN-2 (86%). Also,
TGN-4 and TGN-5 peptides both showed no significant inhibition of
PTEN activity, indicating that PTEN inhibition by TGN-1 and TGN-2
peptides is sequence-specific. In vitro PTEN activity assay using
recombinant PTEN protein and diC8-PIP3 lipid molecule only failed
to show PTEN activity (data not shown).
[0115] IC.sub.50 values for TGN peptides were also measured using
in vitro PTEN activity with TGN peptides in dose-dependent manner
(0.about.100 .mu.M range). The calculated IC.sub.50 values for
TGN-1, TGN-2 and TGN-3 peptides were 19.93 .mu.M, 87.12 .mu.M and
4.83 .mu.M, respectively (FIG. 2C).
Example 2.3--TGN-1 Peptide Promotes PI3K-Akt Signaling Pathway In
Vivo
[0116] The effect of TGN-1 peptide on PI3K signaling pathway in
neuronal cells was determined with PC12 rat pheochromocytoma cell
line. Differentiated PC12 cells, either transfected with PTEN c-DNA
for PTEN overexpression or in the natural state, were incubated
with TGN-1 peptide (10 .mu.M and 100 .mu.M) or TGN-4 peptide (10
.mu.M) at 37.degree. C. for 24 hr. As seen in the diagram in FIG.
3A, if the TGN-1 peptide actually blocks PTEN activity and
suppresses antagonizing effect of PTEN on PI3K activity, the
activation (phosphorylation) level of Akt protein in PI3K signaling
pathway should be increased. Western blot data using anti-phospho
Akt protein antibody showed that the activation (phosphorylation)
level of endogenous Akt protein in PC12 cells treated with TGN-1
peptide increased in TGN-1 peptide dose-dependent manner (FIGS. 3B
and 3C). PC12 cells treated with either TGN-4 peptide or DMSO did
not increase the activation level of AKT protein, suggesting that
promotion of Akt protein phosphorylation level was specifically
triggered by TGN-1 peptide. As the expression level of either
endogenous PTEN (FIG. 3B) or overexpressed PTEN (FIG. 3C) showed no
difference in activity upon treatment with TGN peptides or DMSO, it
is clear that TGN-1 peptide specifically inhibits PTEN activity to
suppress down-regulation effect of PTEN on PI3K signaling pathway
and facilitate PI3K-Akt signaling pathway.
Example 2.4--TGN-1 and TGN-2 Peptides Show Neurotrophic Effects
Including Neuroprotection in Neuronal Cell Culture
[0117] We investigated the effect of TGN peptides against neurite
degeneration on differentiated neuronal cells. Neurite degeneration
was induced in PC12 cells by interfering with the cells' neuritic
microtubule dynamics by contacting the cells with Nocodazole.
Differentiated rat PC12 cells were treated with Nocodazole (0.5
.mu.M) first and incubated with fresh media containing NGF (50
ng/mL) and TGN peptides (100 .mu.M) for 72 hrs. Immunofluorescence
analysis using two different tubulin antibodies (acetylated
.alpha.-tubulin antibody for stable neurites and TUJ-1
.beta.-tubulin antibody for total neurites) demonstrated that TGN-1
and TGN-2 peptides clearly delayed Nocodazole-induced neurite
degeneration via microtubule stabilization (FIG. 4A). We further
investigated the effect of TGN peptides on neurite outgrowth of
PC12 cells. Addition of TGN peptides to the differentiating PC12
cells actually promoted neurite development (2.4-time increment by
TGN-1 and 1.6-time increment by TGN-2, FIG. 4B). Taken together, we
TGN-1 and TGN-2 peptides show neurotrophic effect as well as the
activity of protecting mature neurites from degeneration.
REFERENCES
[0118] Campbell, R. B., Liu, F., and Ross, A. H. "Allosteric
Activation of PTEN Phosphatase by Phosphatidylinositol
4,5-Bisphosphate", J. Biol. Chem., 2003, 278, pp. 33617-33620.
[0119] Christie, K. J., Webber, C. A., Martinez J. A., Singh, B.
and Zochodne, D. W. "PTEN Inhibition to Facilitate Intrinsic
Regenerative Outgrowth of Adult Peripheral Axons.", J. Neuro. Sci.,
2010, 30(27), pp. 9306-9315. [0120] Das, S., Dixon, J. E. and Cho,
W. "Membrane-binding and activation mechanism of PTEN", PNAS, 2003,
100(13), pp. 7491-7496. [0121] Di Cristofano, A. and Pandolfi, P.
P. "The multiple roles of PTEN in tumor suppression", Cell, 2000,
100, (4), pp. 387-390. [0122] Filbin, M. T. "Recapitulate
development to promote axonal regeneration: good or bad approach?",
Philos. Trans. R. Soc. London B Biol. Sci., 2006, 361, pp.
1565-1574. [0123] Fitch, M. T., Silver, J. "CNS injury, glial
scars, and inflammation: Inhibitory extracellular matrices and
regeneration failure.", Exp Neurol., 2008, 209, pp. 294-301. [0124]
Georgescu, M. M., Kirsch, K. H., Kaloudis, P., Yang, H., Pavletich,
N. P. and Hanafusa, H. "Stabilization and productive positioning
roles of the C2 domain of PTEN tumor suppressor.", Cancer Res.,
2000, 60, pp. 7033-7038. [0125] Goldberg, J. L., Klassen, M. P.,
Hua, Y. and Barres, B. A. "Amacrine-Signaled Loss of Intrinsic Axon
Growth Ability by Retinal Ganglion Cells.", Science, 2002, 296, pp.
1860-1864. [0126] Hellal, F. et al. Microtubule stabilization
reduces scarring and causes axon regeneration after spinal cord
injury", Science, 2011; "331, pp. 928-931. [0127] Lee, J. O., Yang,
H., Georgescu, M. M., Di Cristofano, A., Maehama, T., Shi, Y.,
Dixon, J. E., Pandolfi, P., and Pavletich, N. P. "Crystal Structure
of the PTEN Tumor Suppressor: Implications for Its Phosphoinositide
Phosphatase Activity and Membrane Association", Cell, 1999, 99, pp.
323-334. [0128] Leslie, N. R., Batty, I. H., Maccario, H.,
Davidson, L. and Downes, C. P. "Understanding PTEN regulation:
PIP2, polarity and protein stability", Oncogene, 2008, 27, pp.
5464-5476. [0129] Liu, K. et. al. "PTEN deletion enhances the
regenerative ability of adult corticospinal neurons.", Nat.
Neurosci., 2010, 13, pp. 1075-1081. [0130] Miller, S. J., Lou, D.
Y., Seldin, D. C., Lane, W. S., and Neel, B. G. "Direct
identification of PTEN phosphorylation sites.", FEBS Lett., 2002,
528, pp. 145-153. [0131] Molina, J. R., Morales, F. C., Hayashi,
Y., Aldape, K. D. and Georgescu, M-M. "Loss of PTEN Binding Adapter
Protein NHERF1 from Plasma Membrane in Glioblastoma Contributes to
PTEN Inactivation", Cancer Res., 2010, 70(17), pp. 6697-703. [0132]
Odriozola, L., Singh, G., Hoang, T. and Chan, A. M. "Regulation of
PTEN Activity by Its Carboxyl-terminal Autoinhibitory Domain", Jol.
Bio. Sci., 2007, 282(32), pp. 23306-23315. [0133] Okahara, F.,
Ikawa, H., Kanaho, Y., and Maehama, T. "Regulation of PTEN
Phosphorylation and Stability by a Tumor Suppressor Candidate
Protein", J. Biol. Chem., 2004, 279, pp. 45300-45303. [0134] Park,
K. K., et al. "Promoting axon regeneration in the adult CNS by
modulation of the PTEN/mTOR pathway.", Science, 2008, 322, pp.
963-966. [0135] Randar, M., Inoue, T., Meyer, T., Zhang, J.,
Vazquez, F. and Devreotesa, P. N. "A phosphorylation dependent
intramolecular interaction regulates the membrane association and
activity of the tumor suppressor PTEN", PNAS, 2009, 106(2), pp.
480-485. [0136] Saijilafu, Hur E M, Liu C M, Jiao Z, Xu W N, Zhou F
Q. PI3K-GSK3 signaling regulates mammalian axon regeneration by
inducing the expression of Smad1. Nature Communication. 2013;
4:2690. [0137] Schwab, M. E. and Bartholdi, D., "Degeneration and
regeneration of axons in the lesioned spinal cord.", Physiol. Rev.
1996, 76, pp. 319-370. [0138] Sengottuvel V, Fischer D.
Facilitating axon regeneration in the injured CNS by microtubules
stabilization. Commun Integr Biol. 2011; 4:391-3. [0139] Stambolic,
V., Suzuki, A., De la Pompa, J. L. et al. "Negative regulation of
PKB/Akt-dependent cell survival by the tumor suppressor PTEN.",
Cell, 1998, 95(1), pp. 29-39. [0140] Sun, F., Park, K. K., et al.
"Sustained axon regeneration induced by co-deletion of PTEN and
SOCS3", Nature, 2012, 480(7377), pp. 372-375. [0141] Takahashi, Y.,
Morales, F. C., Kreimann, E. L. and Georgescu, G. M. "PTEN tumor
suppressor associates with NHERF proteins to attenuate PDGF
receptor signaling", EMBO J., 2006, 25, pp. 910-920. [0142]
Takemura R, Okabe S, Umeyama T, Kanai Y, Cowan N J, Hirokawa N.
Increased microtubule stability and alpha tubulin acetylation in
cells transfected with microtubule-associated proteins MAP1B, MAP2
or tau. J Cell Sci. 1992; 103:953-964. [0143] Vazquez, F., and
Devreotes, P. "Regulation of PTEN function as a PIPS gatekeeper
through membrane interaction.", Cell Cycle, 2006, 5, pp. 1523-1527.
[0144] Vazquez, F., Grossman, S. R., Takahashi, Y., Rokas, M. V.,
Nakamura, N. and Sellers, W. R. "Phosphorylation of the PTEN Tail
Acts as an Inhibitory Switch by Preventing Its Recruitment into a
Protein Complex", J. Biol. Chem., 2001, 276, pp 48627-48630. [0145]
Walker, S. M., Leslie, N. R., Perera, N. M., Batty, I. H., and
Downes, C. P. "The tumour-suppressor function of PTEN requires an
N-terminal lipid-binding motif", Biochem. J., 2004, 379, pp.
301-307. [0146] Witte H, Neukirchen D, Bradke F, Microtubule
stabilization specifies initial neuronal polarization. J Cell Biol.
2008; 180:619-632. [0147] Yiu, G. and He, Z. "Glial inhibition of
CNS axon regeneration.", Nat. Rev. Neurosci., 2006, 7, pp. 617-627.
[0148] Zhang, S. and Yu, D. "PI(3)King Apart PTEN's Role in
Cancer", Clin Cancer Res, 2010, 16, pp. 4325-4330.
[0149] All of the references cited herein are incorporated by
reference in their entirety.
[0150] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention
specifically described herein.
Sequence CWU 1
1
121403PRTArtificial SequencePTEN amino acid sequence 1Met Thr Ala
Ile Ile Lys Glu Ile Val Ser Arg Asn Lys Arg Arg Tyr1 5 10 15Gln Glu
Asp Gly Phe Asp Leu Asp Leu Thr Tyr Ile Tyr Pro Asn Ile 20 25 30Ile
Ala Met Gly Phe Pro Ala Glu Arg Leu Glu Gly Val Tyr Arg Asn 35 40
45Asn Ile Asp Asp Val Val Arg Phe Leu Asp Ser Lys His Lys Asn His
50 55 60Tyr Lys Ile Tyr Asn Leu Cys Ala Glu Arg His Tyr Asp Thr Ala
Lys65 70 75 80Phe Asn Cys Arg Val Ala Gln Tyr Pro Phe Glu Asp His
Asn Pro Pro 85 90 95Gln Leu Glu Leu Ile Lys Pro Phe Cys Glu Asp Leu
Asp Gln Trp Leu 100 105 110Ser Glu Asp Asp Asn His Val Ala Ala Ile
His Cys Lys Ala Gly Lys 115 120 125Gly Arg Thr Gly Val Met Ile Cys
Ala Tyr Leu Leu His Arg Gly Lys 130 135 140Phe Leu Lys Ala Gln Glu
Ala Leu Asp Phe Tyr Gly Glu Val Arg Thr145 150 155 160Arg Asp Lys
Lys Gly Val Thr Ile Pro Ser Gln Arg Arg Tyr Val Tyr 165 170 175Tyr
Tyr Ser Tyr Leu Leu Lys Asn His Leu Asp Tyr Arg Pro Val Ala 180 185
190Leu Leu Phe His Lys Met Met Phe Glu Thr Ile Pro Met Phe Ser Gly
195 200 205Gly Thr Cys Asn Pro Gln Phe Val Val Cys Gln Leu Lys Val
Lys Ile 210 215 220Tyr Ser Ser Asn Ser Gly Pro Thr Arg Arg Glu Asp
Lys Phe Met Tyr225 230 235 240Phe Glu Phe Pro Gln Pro Leu Pro Val
Cys Gly Asp Ile Lys Val Glu 245 250 255Phe Phe His Lys Gln Asn Lys
Met Leu Lys Lys Asp Lys Met Phe His 260 265 270Phe Trp Val Asn Thr
Phe Phe Ile Pro Gly Pro Glu Glu Thr Ser Glu 275 280 285Lys Val Glu
Asn Gly Ser Leu Cys Asp Gln Glu Ile Asp Ser Ile Cys 290 295 300Ser
Ile Glu Arg Ala Asp Asn Asp Lys Glu Tyr Leu Val Leu Thr Leu305 310
315 320Thr Lys Asn Asp Leu Asp Lys Ala Asn Lys Asp Lys Ala Asn Arg
Tyr 325 330 335Phe Ser Pro Asn Phe Lys Val Lys Leu Tyr Phe Thr Lys
Thr Val Glu 340 345 350Glu Pro Ser Asn Pro Glu Ala Ser Ser Ser Thr
Ser Val Thr Pro Asp 355 360 365Val Ser Asp Asn Glu Pro Asp His Tyr
Arg Tyr Ser Asp Thr Thr Asp 370 375 380Ser Asp Pro Glu Asn Glu Pro
Phe Asp Glu Asp Gln His Thr Gln Ile385 390 395 400Thr Lys
Val28PRTArtificial SequencePTD (peptide transfer domain) 2Arg Arg
Arg Arg Arg Arg Arg Arg1 5327PRTArtificial SequenceTGN-1 peptide
3Val Thr Pro Asp Val Pro Ser Asp Asn Glu Pro Asp His Tyr Arg Tyr1 5
10 15Pro Ser Asp Thr Thr Asp Pro Ser Asp Pro Glu 20
25435PRTArtificial SequencePTD and TGN-1 4Arg Arg Arg Arg Arg Arg
Arg Arg Val Thr Pro Asp Val Pro Ser Asp1 5 10 15Asn Glu Pro Asp His
Tyr Arg Tyr Pro Ser Asp Thr Thr Asp Pro Ser 20 25 30Asp Pro Glu
35530PRTArtificial SequencePTD and TGN-2 5His Tyr Arg Tyr Pro Ser
Asp Thr Thr Asp Pro Ser Asp Pro Glu Asn1 5 10 15Glu Pro Phe Asp Glu
Asp Gln His Thr Gln Ile Thr Lys Val 20 25 30638PRTArtificial
SequencePTD and TGN-2 6Arg Arg Arg Arg Arg Arg Arg Arg His Tyr Arg
Tyr Pro Ser Asp Thr1 5 10 15Thr Asp Pro Ser Asp Pro Glu Asn Glu Pro
Phe Asp Glu Asp Gln His 20 25 30Thr Gln Ile Thr Lys Val
35728PRTArtificial SequencePTD and TGN-3 7His Tyr Arg Tyr Val Asp
Thr Thr Asp Val Asp Pro Glu Asn Glu Pro1 5 10 15Phe Asp Glu Asp Gln
His Thr Gln Ile Thr Lys Val 20 25836PRTArtificial SequencePTD and
TGN-3 8Arg Arg Arg Arg Arg Arg Arg Arg His Tyr Arg Tyr Val Asp Thr
Thr1 5 10 15Asp Val Asp Pro Glu Asn Glu Pro Phe Asp Glu Asp Gln His
Thr Gln 20 25 30Ile Thr Lys Val 35923PRTArtificial SequencePTD and
TGN-4 9Ser Asp Asp Glu Tyr Thr Asp Asn Pro Asp Ser Arg Tyr Val Ser
Asp1 5 10 15Thr Pro Val Asp Thr Glu His 201031PRTArtificial
SequencePTD and TGN-4 10Arg Arg Arg Arg Arg Arg Arg Arg Ser Asp Asp
Glu Tyr Thr Asp Asn1 5 10 15Pro Asp Ser Arg Tyr Val Ser Asp Thr Pro
Val Asp Thr Glu His 20 25 301128PRTArtificial SequencePTD and TGN-5
11Asp Glu His Asp Thr Glu Tyr Thr Pro Asp Tyr Arg Gln Glu Thr His1
5 10 15Phe Asn Ser Gln Pro Thr Asp Lys Ser Asp Val Ile 20
251236PRTArtificial SequencePTD attached at the N-terminus of TGN-5
12Arg Arg Arg Arg Arg Arg Arg Arg Asp Glu His Asp Thr Glu Tyr Thr1
5 10 15Pro Asp Tyr Arg Gln Glu Thr His Phe Asn Ser Gln Pro Thr Asp
Lys 20 25 30Ser Asp Val Ile 35
* * * * *