U.S. patent application number 14/993116 was filed with the patent office on 2016-05-12 for glycogen synthase kinase-3 inhibitors.
This patent application is currently assigned to Ramot at Tel-Aviv University Ltd.. The applicant listed for this patent is Ramot at Tel-Aviv University Ltd.. Invention is credited to Hagit Eldar-Finkelman, Avital Licht-Murava, Batya Plotkin.
Application Number | 20160130303 14/993116 |
Document ID | / |
Family ID | 45607317 |
Filed Date | 2016-05-12 |
United States Patent
Application |
20160130303 |
Kind Code |
A1 |
Eldar-Finkelman; Hagit ; et
al. |
May 12, 2016 |
GLYCOGEN SYNTHASE KINASE-3 INHIBITORS
Abstract
Novel peptide inhibitors of GSK-3, compositions containing same
and uses thereof are disclosed. The novel peptide inhibitors are
substrate-competitive inhibitors and have an amino acid sequence
designed so as to bind to a defined binding site subunit in GSK-3.
Also disclosed are GSK-3 substrate competitive inhibitors which
bind to the defined binding site subunit in the enzyme. Also
disclosed are mutants of GSK-3 and uses thereof for identifying a
putative GSK-3 substrate competitive inhibitor.
Inventors: |
Eldar-Finkelman; Hagit;
(Shoham, IL) ; Licht-Murava; Avital; (Tel-Aviv,
IL) ; Plotkin; Batya; (Rishon-LeZion, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ramot at Tel-Aviv University Ltd. |
Tel-Aviv |
|
IL |
|
|
Assignee: |
Ramot at Tel-Aviv University
Ltd.
Tel-Aviv
IL
|
Family ID: |
45607317 |
Appl. No.: |
14/993116 |
Filed: |
January 12, 2016 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13981668 |
Jul 25, 2013 |
9243034 |
|
|
PCT/IB2012/050373 |
Jan 26, 2012 |
|
|
|
14993116 |
|
|
|
|
61436640 |
Jan 27, 2011 |
|
|
|
Current U.S.
Class: |
514/21.5 ;
435/184; 506/8; 506/9; 530/327 |
Current CPC
Class: |
G16B 35/00 20190201;
G01N 2500/10 20130101; C12N 9/1205 20130101; G16C 20/60 20190201;
C07K 7/06 20130101; G01N 2500/04 20130101; A61K 38/08 20130101;
A61K 38/00 20130101; A61K 38/10 20130101; C12Q 1/485 20130101; C12N
9/12 20130101; G01N 33/573 20130101; C07K 7/08 20130101; G01N
2333/912 20130101; C12Y 207/11001 20130101; G01N 2440/14
20130101 |
International
Class: |
C07K 7/08 20060101
C07K007/08; C40B 30/02 20060101 C40B030/02; G01N 33/573 20060101
G01N033/573 |
Claims
1. A peptide having the amino acid sequence I: [Yn . . .
Y.sub.1]ZX.sub.1X.sub.2X.sub.3S(p)[W.sub.1 . . . Wm] (I) wherein, m
equals 1 or 2; n is 3, 4, 5, 6 or 7, such that said peptide
consists of 10 to 13 amino acid residues; S(p) is a phosphorylated
serine residue or a phosphorylated threonine residue; Z is any
amino acid residue excepting serine residue or threonine residue;
X.sub.1, X.sub.2, Y.sub.1-Yn and W.sub.1-Wm are each independently
any amino acid residue; and X.sub.3 is a hydrophobic amino acid
residue.
2. The peptide of claim 1, wherein X.sub.3 is selected from the
group consisting of a proline residue and an alanine residue.
3. The peptide of claim 1, wherein X.sub.3 is a proline
residue.
4. The peptide of claim 1, wherein each of X.sub.1, X.sub.2 and
X.sub.3 is a hydrophobic amino acid residue.
5. The peptide of claim 4, wherein each of X.sub.1, X.sub.2 and
X.sub.3 is independently selected from the group consisting of a
proline residue and an alanine residue.
6. The peptide of claim 5, wherein X.sub.1 and X.sub.2 are each a
proline residue.
7. The peptide of claim 1, wherein S(p) is a phosphorylated
serine.
8. The peptide of claim 1, wherein Z is an alanine residue.
9. The peptide of claim 1, wherein m is 1 and W.sub.1 is a proline
residue.
10. The peptide of claim 1, wherein n is 5.
11. The peptide of claim 10, wherein Y.sub.1-Y.sub.5 has the amino
acid sequence Lys-Glu-Ala-Pro-Pro.
12. The peptide of claim 1, having an amino acid sequence selected
from the group of amino acid sequences as set forth in SEQ ID
NOS:11-13 and 16.
13. The peptide of claim 1, consisting of the amino acid sequence
as set forth in SEQ ID NO:16.
14. The peptide of claim 1, further comprising a hydrophobic moiety
attached thereto.
15. The peptide of claim 14, wherein said hydrophobic moiety is
selected from the group consisting of a fatty acid and a fatty acid
attached to an amino acid residue.
16. The peptide of claim 15, wherein said fatty acid is myristic
acid.
17. The peptide of claim 16, consisting of the amino acid sequence
as set forth in SEQ ID NO:17.
18. A pharmaceutical composition comprising, as an active
ingredient, the peptide of claim 1, and a pharmaceutically
acceptable carrier.
19. The pharmaceutical composition of claim 18, being packaged in a
packaging material and identified in print, on or in said packaging
material, for use in inhibiting an activity of GSK-3.
20. The pharmaceutical composition of claim 18, being packaged in a
packaging material and identified in print, on or in said packaging
material, for use in the treatment of a biological condition
associated with GSK-3 activity.
21. A pharmaceutical composition comprising, as an active
ingredient, the peptide of claim 14, and a pharmaceutically
acceptable carrier.
22. A method of inhibiting an activity of GSK-3, the method
comprising contacting cells expressing GSK-3 with an effective
amount of the peptide of claim 1.
23. A method of treating a biological condition associated with
GSK-3 activity, the method comprising administering to a subject in
need thereof a therapeutically effective amount of the peptide of
claim 1.
24. A method of inhibiting an activity of GSK-3, the method
comprising contacting cells expressing GSK-3 with an effective
amount of the peptide of claim 14.
25. A method of treating a biological condition associated with
GSK-3 activity, the method comprising administering to a subject in
need thereof a therapeutically effective amount of the peptide of
claim 14.
26. A GSK-3 substrate competitive inhibitor capable of interacting
with at least one amino acid within the catalytic binding site of a
GSK-3 enzyme, said at least one amino acid comprising a
phenylalanine residue at position 93, or an equivalent thereof of
said GSK-3 enzyme.
27. A method of identifying a putative substrate competitive
inhibitor of GSK-3, the method comprising screening a plurality of
substances for a substance capable of interacting with a
phenylalanine residue at position 93, or an equivalent thereof,
within a catalytic binding site of GSK-3.
28. The method of claim 27, comprising screening said plurality of
substances for a substance which exhibits inhibition of at least
20% of an activity of a wild-type GSK-3 enzyme and which exhibits
inhibition of less than 20% of said activity of a mutated GSK-3
enzyme, said mutated GSK-3 enzyme comprising an amino acid
substitution with respect to position Phe93, or an equivalent
thereof, of a corresponding wild-type GSK3 enzyme.
29. The method of claim 28, wherein said screening comprises:
determining said activity of said wild-type GSK-3 enzyme in the
presence and absence of each of said substances, thereby
determining a level of inhibition of said activity of said
wild-type GSK-3 enzyme exhibited by each of said substances;
determining said activity of said mutated GSK-3 enzyme in the
presence and absence of each of said substances, thereby
determining a level of inhibition of said activity of said mutated
GSK-3 enzyme exhibited by each of said substances; and comparing
said levels of inhibition.
30. The method of claim 27, wherein said screening comprises
computationally screening said plurality of substances for a
substance capable of interacting with a phenylalanine residue at
position 93, or an equivalent amino acid thereof, within a set of
atomic structural coordinates defining a three-dimensional atomic
structure of a GSK-3 enzyme.
31. An isolated polypeptide comprising an amino acid sequence of a
mutated GSK-3 enzyme, wherein an amino acid sequence of said
mutated GSK-3 enzyme comprises at least one amino acid substitution
with respect to position Asp90, Lys91, Arg92, Phe93 and/or Lys94 of
a corresponding wild-type GSK3 enzyme.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/981,668 filed on Jul. 25, 2013, which is a
National Phase of PCT Patent Application No. PCT/IB2012/050373
having International filing date of Jan. 26, 2012, which claims the
benefit of priority under 35 USC .sctn.119(e) of U.S. Provisional
Patent Application No. 61/436,640 filed on Jan. 27, 2011. The
contents of the above applications are all incorporated by
reference as if fully set forth herein in their entirety.
SEQUENCE LISTING STATEMENT
[0002] The ASCII file, entitled 64746SequenceListing.txt, created
on Dec. 6, 2015, comprising 40,128 bytes, submitted concurrently
with the filing of this application is incorporated herein by
reference.
FIELD AND BACKGROUND OF THE INVENTION
[0003] The present invention, in some embodiments thereof, relates
to novel glycogen synthase kinase-3 (GSK-3) inhibitors and, more
particularly, but not exclusively, to novel substrate-competitive
inhibitors of glycogen synthase kinase-3 (GSK-3) and to the use of
such inhibitors in the treatment of biological conditions
associated with GSK-3 activity.
[0004] Protein kinases and phosphorylation cascades are essential
for life and play key roles in the regulation of many cellular
processes including cell proliferation, cell cycle progression,
metabolic homeostasis, transcriptional activation and development.
Aberrant regulation of protein phosphorylation underlies many human
diseases, and this has prompted the development and design of
protein kinase inhibitors. Most of the protein kinase inhibitors
developed so far compete with ATP for its binding site. These
inhibitors, although often very effective, generally show limited
specificity due to the fact that the ATP binding site is highly
conserved among protein kinases.
[0005] Other sites, such as the substrate's binding site, show more
variability in their shape and amino acid compositions and may
serve as favorable sites for drug design. Understanding of
substrate recognition and specificity is thus essential for
development of substrate competitive inhibitors. This knowledge,
however, is limited by the scarce amount of structural data
regarding substrate binding.
[0006] Glycogen synthase kinase-3 (GSK-3) is a constitutively
active serine/threonine kinase that modulates diverse cellular
functions including metabolism, cell survival and migration,
neuronal signaling and embryonic development. Deregulation of GSK-3
activity has been implicated in the pathogenesis of human diseases
such as, for example, type-2 diabetes, neurodegenerative disorders
and psychiatric disorders. Selective inhibition of GSK-3 is thought
to be of therapeutic value in treating these disorders [Bhat et al.
(2004). J. Neurochem. 89, 1313-7; Cohen, P. & Goedert, M.
(2004). Nat. Rev. Drug Discov. 3, 479-87; Meijer et al. (2004)
Trends Pharmacol Sci 25, 471-80; Eldar-Finkelman et al. Biochim
Biophys Acta 1804, 598-603; Martinez, A. & Perez, D. I. (2008)
J. Alzheimers Dis. 15, 181-91].
[0007] Recently, it has been found that GSK-3 is also involved in
the pathogenesis of cardiovascular diseases [Cheng et al. 2010 J.
Mol Cell Cardiol, in press; Kerkela et al. 2008, J. Clin. Invest.
118:3609-18], of malaria and trypanosomiasis [Droucheau et al.
2004, BBRC, 1700:139-140; Ojo et al. 2008, Antimicrob Agents
Chemother, 37107-3717], and in stem cell maintenance or
differentiation [Wray et al. 2010 Biochem Soc Trans 1027-32].
[0008] In view of the wide implication of GSK-3 in various
signaling pathways, development of specific inhibitors for GSK-3 is
considered both promising and important regarding various
therapeutic interventions as well as basic research.
[0009] Some mood stabilizers were found to inhibit GSK-3. However,
while the inhibition of GSK-3 both by lithium chloride (LiCl) (WO
97/41854) and by purine inhibitors (WO 98/16528) has been reported,
these inhibitors are not specific for GSK-3. In fact, it was shown
that these drugs affect multiple signaling pathways, and inhibit
other cellular targets, such as inositol monophosphatase (IMpase)
and histone deacetylases.
[0010] Similarly, an engineered cAMP response element binding
protein (CREB), a known substrate of GSK-3, has been described
(Fiol et al, 1994), along with other potential GSK-3 peptide
inhibitors (Fiol et al, 1990). However, these substrates also only
nominally inhibit GSK-3 activity.
[0011] Other GSK-3 inhibitors have been reported. Two structurally
related small molecules SB-216763 and SB-415286 (GlaxoSmithKline
Pharmaceutical) that specifically inhibited GSK-3 were developed
and were shown to modulate glycogen metabolism and gene
transcription as well as to protect against neuronal death induced
by reduction in PI3 kinase activity (Cross et al., 2001; Coghlan et
al., 2000). Another study indicated that Induribin, the active
ingredient of the traditional Chinese medicine for chronic
myelocytic leukemia, is a GSK-3 inhibitor. However, Indirubin also
inhibits cyclic-dependent protein kinase-2 (CDK-2) (Damiens et al.,
2001). These GSK-3 inhibitors are ATP competitive and were
identified by high throughput screening of chemical libraries. It
is generally accepted that a major drawback of ATP-competitive
inhibitors is their limited specificity (see, for example, Davies
et al., 2000).
[0012] The present inventors have previously reported of a novel
class of substrate competitive inhibitors for GSK-3 [Plotkin et al.
(2003) J. Pharmacol. Exp. Ther., 974-980], designed based on the
unique substrate-recognition motif of GSK-3 that includes a
phosphorylated residue (usually serine) in the context of SXXXS(p)
(where S is the target serine, S(p) is phosphorylate serine and X
is any amino acid) [see also Woodgett & Cohen (1984) Biochim.
Biophys. Acta. 788, 339-47; Fiol et al. (1987) J. Biol. Chem. 262,
14042-8]. Structural studies of GSK-3.beta. identified a likely
docking site for the phosphorylated residue; it is a positively
charged binding pocket composed of Arg96, Arg180, and Lys205
[Dajani et al. (2001) Cell 105, 721-32; ter Haar et al. (2001)
Nature Structural Biology 8, 593-6].
[0013] The short phosphorylated peptides patterned after the GSK-3
substrates behaved as substrate competitive inhibitors (Plotkin et
al., 2003, supra), with the L803 peptide, KEAPPAPPQS(p)P (SEQ ID
NO:4), derived from the substrate heat shock factor-1 (HSF-1)
showing the best inhibition activity of those evaluated. An
advanced version of L803, the cell permeable peptide L803-mts, was
shown to promote beneficial biological activities in conditions
associated with diabetes, neuron growth and survival, and mood
behavior [Kaidanovich-Beilin & Eldar-Finkelman (2005) J.
Pharmacol. Exp. Ther. 316:17-24; Rao et al. (2007) Diabetologia 50,
452-60; Kim et al. (2006) Neuron 52, 981-96; Chen et al. (2004)
Faseb J 18, 1162-4; Kaidanovich-Beilin et al. (2004) Biol.
Psychiatry. 55:781-4; Shapira et al. (2007) Mol. Cell Neurosci. 34,
571-7].
[0014] While further focusing on substrate recognition of GSK-3,
three positions in the vicinity of the catalytic site (Phe67 in the
P-loop, Gln89 and Asn95) were identified as important for GSK-3
substrates binding [Ilouz et al. (2006) J. Biol. Chem. 281,
30621-30].
[0015] Additional background art includes U.S. Pat. Nos. 6,780,625
and 7,378,432; WO 2004/052404 and WO 2005/000192; WO 01/49709;
Liberman, Z. & Eldar-Finkelman, H. (2005) J. Biol. Chem. 280,
4422-8; Liberman et al. (2008) Am. J. Physiol. Endocrinol. Metab.
294, E1169-77; Bertrand et al. (2003) J. Mol. Biol. 333, 393-407;
Licht-Murava et al., J. Mol. Biol. (2011) 408, 366-378; and Palomo
et al. J. Med. Chem. (2012) as published on wwwdotpubsdotacsdotorg
as "Just Accepted Manuscript" on Jan. 18, 2012.
SUMMARY OF THE INVENTION
[0016] According to an aspect of some embodiments of the present
invention there is provided a peptide having the amino acid
sequence I:
[Yn . . . Y.sub.1]ZX.sub.1X.sub.2X.sub.3S(p)[W.sub.1 . . . Wm] (I)
[0017] wherein, [0018] m equals 1 or 2; [0019] n is 3, 4, 5, 6 or
7, such that the peptide consists of 10 to 13 amino acid residues;
[0020] S(p) is a phosphorylated serine residue or a phosphorylated
threonine residue; [0021] Z is any amino acid residue excepting
serine residue or threonine residue;
[0022] X.sub.1, X.sub.2, Y.sub.1-Yn and W.sub.1-Wm are each
independently any amino acid residue; and X.sub.3 is a hydrophobic
amino acid residue.
[0023] According to some embodiments of the present invention,
X.sub.3 is selected from the group consisting of a proline residue
and an alanine residue.
[0024] According to some embodiments of the present invention,
X.sub.3 is a proline residue.
[0025] According to some embodiments of the present invention, each
of X.sub.1, X.sub.2 and X.sub.3 is a hydrophobic amino acid
residue.
[0026] According to some embodiments of the present invention, each
of X.sub.1, X.sub.2 and X.sub.3 is independently selected from the
group consisting of a proline residue and an alanine residue.
[0027] According to some embodiments of the present invention,
X.sub.1 and X.sub.2 are each a proline residue.
[0028] According to some embodiments of the present invention, S(p)
is a phosphorylated serine.
[0029] According to some embodiments of the present invention, Z is
an alanine residue.
[0030] According to some embodiments of the present invention, m is
1 and W.sub.1 is a proline residue.
[0031] According to some embodiments of the present invention, n is
5.
[0032] According to some embodiments of the present invention,
Y.sub.1--Y.sub.5 has the amino acid sequence Lys-Glu-Ala-Pro-Pro
(SEQ ID NO:48).
[0033] According to some embodiments of the present invention, the
peptide has an amino acid sequence selected from the group of amino
acid sequences as set forth in SEQ ID NOS:11-13 and 16.
[0034] According to some embodiments of the present invention, the
peptide is consisting of the amino acid sequence as set forth in
SEQ ID NO:16.
[0035] According to some embodiments of the present invention, any
of the peptides described herein further comprises a hydrophobic
moiety attached thereto.
[0036] According to some embodiments of the present invention, the
hydrophobic moiety is selected from the group consisting of a fatty
acid and a fatty acid attached to an amino acid residue.
[0037] According to some embodiments of the present invention, the
fatty acid is myristic acid.
[0038] According to some embodiments of the present invention, the
peptide consists of the amino acid sequence as set forth in SEQ ID
NO:17.
[0039] According to an aspect of some embodiments of the present
invention there is provided a pharmaceutical composition
comprising, as an active ingredient, the peptide as described
herein, and a pharmaceutically acceptable carrier.
[0040] According to some embodiments of the present invention, the
pharmaceutical is packaged in a packaging material and identified
in print, on or in the packaging material, for use in inhibiting an
activity of GSK-3.
[0041] According to some embodiments of the present invention, the
pharmaceutical composition is packaged in a packaging material and
identified in print, on or in the packaging material, for use in
the treatment of a biological condition associated with GSK-3
activity.
[0042] According to an aspect of some embodiments of the present
invention there is provided a peptide as described herein, for use
in inhibiting an activity of GSK-3.
[0043] According to an aspect of some embodiments of the present
invention there is provided a peptide as described herein for use
in the treatment of a biological condition associated with GSK-3
activity.
[0044] According to an aspect of some embodiments of the present
invention there is provided a method of inhibiting an activity of
GSK-3, the method comprising contacting cells expressing GSK-3 with
an effective amount of the peptide as described herein.
[0045] According to an aspect of some embodiments of the present
invention there is provided a use of the peptide as described
herein in the manufacture of a medicament for inhibiting an
activity of GSK-3 activity.
[0046] According to some embodiments of the invention, the activity
is a phosphorylation activity and/or an autophosphorylation
activity.
[0047] According to an aspect of some embodiments of the present
invention there is provided a method of treating a biological
condition associated with GSK-3 activity, the method comprising
administering to a subject in need thereof a therapeutically
effective amount of the peptide as described herein.
[0048] According to an aspect of some embodiments of the present
invention there is provided a use of the peptide as described
herein in the manufacture of a medicament for treating a biological
condition associated with GSK-3 activity.
[0049] According to some embodiments of the invention, the
biological condition is associated with overexpression of
GSK-3.
[0050] According to some embodiments of the invention, the
biological condition is selected from the group consisting of
obesity, non-insulin dependent diabetes mellitus, an
insulin-dependent condition, an affective disorder, a
neurodegenerative disease or disorder, a psychotic disease or
disorder, a cardiovascular disease or disorder, a condition
associated with a pathogenic parasite, and a condition treatable by
stem cell transplantation.
[0051] According to an aspect of some embodiments of the present
invention there is provided a GSK-3 substrate competitive inhibitor
capable of interacting with at least one amino acid within the
catalytic binding site of a GSK-3 enzyme, the at least one amino
acid comprising a phenylalanine residue at position 93, or an
equivalent thereof of the GSK-3 enzyme.
[0052] According to some embodiments of the invention, the GSK-3
inhibitor is capable of interacting with at least one additional
amino acid within the catalytic binding site of a GSK-3 enzyme.
[0053] According to some embodiments of the invention, the GSK-3
inhibitor is selected from the group consisting of a peptide, a
polypeptide and an organic small molecule.
[0054] According to some embodiments of the invention, the
screening comprises computationally screening the plurality of
substances for a substance capable of interacting with a
phenylalanine residue at position 93, or an equivalent amino acid
thereof, within a set of atomic structural coordinates defining a
three-dimensional atomic structure of a GSK-3 enzyme.
[0055] According to some embodiments of the invention, the method
is comprising screening the plurality of substances for a substance
which exhibits inhibition of at least 20% of an activity of a
wild-type GSK-3 enzyme and which exhibits inhibition of less than
20% of the activity of a mutated GSK-3 enzyme, the mutated GSK-3
enzyme comprising an amino acid substitution with respect to
position Phe93, or an equivalent thereof, of a corresponding
wild-type GSK3 enzyme.
[0056] According to some embodiments of the invention, the
screening comprises:
[0057] determining the activity of the wild-type GSK-3 enzyme in
the presence and absence of each of the substances, thereby
determining a level of inhibition of the activity of the wild-type
GSK-3 enzyme exhibited by each of the substances;
[0058] determining the activity of the mutated GSK-3 enzyme in the
presence and absence of each of the substances, thereby determining
a level of inhibition of the activity of the mutated GSK-3 enzyme
exhibited by each of the substances; and
[0059] comparing the levels of inhibition.
[0060] According to some embodiments of the invention, the activity
is phosphorylation.
[0061] According to some embodiments of the invention, the
screening comprises computationally screening the plurality of
substances for a substance capable of interacting with a
phenylalanine residue at position 93, or an equivalent amino acid
thereof, within a set of atomic structural coordinates defining a
three-dimensional atomic structure of a GSK-3 enzyme.
[0062] According to some embodiments of the invention, the
computationally screening is for a substance that is further
capable of interacting with at least one additional amino acid
within the catalytic binding site of the GSK-3.
[0063] According to an aspect of some embodiments of the present
invention there is provided an isolated polypeptide comprising an
amino acid sequence of a mutated GSK-3 enzyme, wherein an amino
acid sequence of the mutated GSK-3 enzyme comprises at least one
amino acid substitution with respect to position Asp90, Lys91,
Arg92, Phe93 and/or Lys94 of a corresponding wild-type GSK3
enzyme.
[0064] According to some embodiments of the invention, theta least
one amino acid substitution is with respect to position Asp90,
Arg92, Phe93 and/or Lys94 of the corresponding wild-type GSK-3.
[0065] According to some embodiments of the invention, theta least
one amino acid substitution is with respect to position Phe93 of
the corresponding wild-type GSK-3.
[0066] According to some embodiments of the invention, the amino
acid substitution comprises an alanine substitution.
[0067] According to some embodiments of the invention, the mutated
GSK-3 enzyme comprises an amino acid sequence selected from the
group consisting of the amino acid sequences set forth in SEQ ID
NOS:6-10.
[0068] According to some embodiments of the invention, the mutated
GSK-3 enzyme comprises an amino acid sequence as set forth in SEQ
ID NO:9.
[0069] According to an aspect of some embodiments of the present
invention there is provided a polynucleotide encoding the
polypeptide of any of claims 39-44.
[0070] According to an aspect of some embodiments of the present
invention there is provided a nucleic acid construct comprising the
polynucleotide of claim 45.
[0071] According to an aspect of some embodiments of the present
invention there is provided a host cell system comprising the
nucleic acid construct of claim 46.
[0072] According to an aspect of some embodiments of the present
invention there is provided a method of identifying a putative GS
K-3 substrate competitive inhibitor, the method comprising
screening a plurality of substances for a substance which exhibits
inhibition of at least 20% of an activity of a wild-type GSK-3
enzyme and which exhibits inhibition of no more than 20% of the
activity of any of the mutated GSK-3 enzymes comprised in the
polypeptide as described herein.
[0073] According to some embodiments of the invention, the
screening comprises:
[0074] determining the activity of the wild-type GSK-3 enzyme in
the presence and absence of each of the substances, thereby
determining a level of inhibition of the activity of the wild-type
GSK-3 enzyme exhibited by each of the substances;
[0075] determining the activity of the mutated GSK-3 enzyme in the
presence and absence of each of the substances, thereby determining
a level of inhibition of the activity of the mutated GSK-3 enzyme
exhibited by each of the substances; and comparing the levels of
inhibition.
[0076] According to some embodiments of the invention, the activity
is phosphorylation.
[0077] Unless otherwise defined, all technical and/or scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which the invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of
embodiments of the invention, exemplary methods and/or materials
are described below. In case of conflict, the patent specification,
including definitions, will control. In addition, the materials,
methods, and examples are illustrative only and are not intended to
be necessarily limiting.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0078] Some embodiments of the invention are herein described, by
way of example only, with reference to the accompanying drawings.
With specific reference now to the drawings in detail, it is
stressed that the particulars shown are by way of example and for
purposes of illustrative discussion of embodiments of the
invention. In this regard, the description taken with the drawings
makes apparent to those skilled in the art how embodiments of the
invention may be practiced.
[0079] In the drawings:
[0080] FIGS. 1A-1G present the modifications made to the 88-97
binding subsite of GSK-3.beta. (SEQ ID NO:49) which includes the
89-95 loop (SEQ ID NO:2) (FIG. 1A), Western Blot analyses showing
the expression of GSK-3.beta. mutants (FIG. 1B), a bar graph
showing the phosphorylation of GSK-3 substrates by the F93A mutant
(FIG. 1C); Western Blot analysis showing the expression levels of
CREB and GSK-3 proteins and the phosphorylation of CREB by the F93A
mutant in cells (FIG. 1D); a bar graph presenting the ratio of
pCREB/CREB in cells expressing the F93A mutant (FIG. 1E); Western
Blot analysis showing the expression levels of N'IRS and GSK-3
proteins and the phosphorylation of N'IRS substrate by the F93A
mutant in cells (FIG. 1F); and a bar graph presenting the ratio of
PN'IRS/N'IRS in cells expressing the F93A mutant (FIG. 1G);
[0081] FIGS. 2A-2C are bar graphs showing that L803-mts is a
substrate competitive inhibitor of purified GSK-3.beta. (FIG. 2A)
and that both L803-mts and L803 do not inhibit substrate
phosphorylation by F93A (FIGS. 2B and 2C, respectively);
[0082] FIGS. 3A-3C show the exemplary L803 (SEQ ID NO:4) variants
PK1A (SEQ ID NO:11), PE2A (SEQ ID NO:12), PQ9A (SEQ ID NO:13), PQ9R
(SEQ ID NO:14) and PQ9Y (SEQ ID NO:15) according to some
embodiments of the present invention (FIG. 3A) and the ability of
these variants (250 .mu.M each) to inhibit GSK-3.beta. (FIGS. 3A
and 3B);
[0083] FIG. 4 is a bar graph showing inhibition of GSK-3.beta. by
PQ9P (SEQ ID NO:16);
[0084] FIG. 5 presents dose-response comparative plots showing the
ability of L803 (SEQ ID NO:4), PQ9A (SEQ ID NO:13) and PQ9P (SEQ ID
NO:16) to inhibit GSK-3.beta. at indicated concentrations
(Substrate phosphorylation obtained in reaction with no inhibitor
was defined as 100%, and results presented are means of two
independent experiments each performed in duplicate.+-.SEM);
[0085] FIGS. 6A-6C present dose-response comparative plots showing
the ability of L803-mts and L806-mts to inhibit GSK-3.beta. at
indicated concentrations (Substrate phosphorylation obtained in
reaction with no inhibitor was defined as 100%, and results
presented are means of two independent experiments each performed
in duplicate.+-.SEM) (FIG. 6A), a Western Blot analysis showing the
levels of .beta.-catenin in cells treated with L806-mts, and a
Western Blot analysis showing the phosphorylation of the GSK-3
substrate HSF-1 in COS-7 cells treated with L806-mts; and
[0086] FIGS. 7A-7B present Western Blot analyses showing the
expression level of GSK-3.beta. and the inhibition of GSK-3,
expressed as hippocampus .beta.-catenin levels in mice treated
intranasally with L803-mts (FIG. 7A) and L806-mts (FIG. 7B) and in
non-treated mice (Con).
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
[0087] The present invention, in some embodiments thereof, relates
to novel glycogen synthase kinase-3 (GSK-3) inhibitors and, more
particularly, but not exclusively, to novel substrate-competitive
peptide inhibitors of glycogen synthase kinase-3 (GSK-3) and to the
use of such peptide inhibitors in the treatment of biological
conditions associated with GSK-3 activity.
[0088] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not
necessarily limited in its application to the details set forth in
the following description or exemplified by the Examples. The
invention is capable of other embodiments or of being practiced or
carried out in various ways.
[0089] The present inventors have previously described that
peptides designed after the recognition motif of a GSK-3 substrate
are useful as GSK-3 substrate competitive inhibitors. See, for
example, WO 01/49709 and U.S. Patent Application No. 20020147146,
which are incorporated by reference as if fully set forth
herein.
[0090] These peptides were designed further to the findings that
GSK-3 has a unique recognition motif, and thus that short peptides
which are designed with reference to this motif are highly specific
GSK-3 inhibitors.
[0091] The unique recognition motif of GSK-3, as set forth in SEQ
ID NO:3, is SX.sub.1X.sub.2X.sub.3S(p), where S is serine or
threonine, each of X.sub.1, X.sub.2 and X.sub.3 is any amino acid,
and S(p) is phosphorylated serine or phosphorylated threonine.
Based on this recognition motif, a set of peptides, which differ
one from another in various parameters (e.g., length,
phosphorylation, sequence, etc.) have been designed, synthesized
and were tested for their activity as either substrates or
inhibitors of GSK-3.
[0092] Based on these experiments, a number of features, which
would render a peptide an efficient GSK-3 inhibitor, have been
determined. For example, it was found that the phosphorylated
serine or threonine residue in the motif is necessary for binding.
Without this residue, the peptide will neither be a substrate nor
an inhibitor. It was further determined that a serine (or
threonine) residue upstream of the phosphorylated serine (or
threonine) residue separated by three additional residues renders
the peptide a GSK-3 substrate, whereas replacement of this serine
or threonine residue by any other amino acid, preferably alanine,
converts the substrate to a GSK-3 inhibitor. It was further found
that the number of the additional residues, outside the recognition
motif, affect the inhibition potency of the peptide, such that, for
example, a total number of between 7 and 50, preferably, between 7
and 20, more preferably between 10 and 13 amino acid residues, is
preferable.
[0093] Hence, it was previously described that peptides having the
general amino acid sequence denoted herein as general sequence
I*:
[Yn . . . Y.sub.1]ZX.sub.1X.sub.2X.sub.3S(p)[W.sub.1 . . . Wm]
(I*)
wherein m equals 1 or 2; n is an integer from 1 to 50; S(p) is a
phosphorylated serine residue or a phosphorylated threonine
residue; Z is any amino acid residue excepting serine residue or
threonine residue; and X.sub.1, X.sub.2, X.sub.3, Y.sub.1-Yn and
W.sub.1-Wm are each independently any amino acid residue, are
highly efficient and specific inhibitors of GSK-3. See, for
example, U.S. Pat. Nos. 6,780,625 and 7,378,432; WO 2004/052404 and
WO 2005/000192; and WO 01/49709, which are incorporated by
reference as if fully set forth herein. It is noted that since
these previously described inhibitors were designed so as to modify
an amino acid sequence of known GSK-3 substrates, the nature of the
amino acid residues presented by variables X.sub.1, X.sub.2,
X.sub.3, Y.sub.1-Yn and W.sub.1-Wm in the amino acid sequence I*
was typically defined per the corresponding residues in the known
GSK-3 substrate, namely, X.sub.1, X.sub.2, X.sub.3 were the same as
correspond amino acid residues between a serine and a
phosphorylated serine in a known GSK-3 substrate, Y.sub.1-Yn were
the same as the amino acid residues upstream the serine residue,
and W.sub.1-Wm were the same as the amino acid residues downstream
the phosphorylated serine or threonine residue of a known GSK-3
substrate.
[0094] It was further described that preferred peptides are those
having an alanine residue at the Z position, having any amino acid
residue excepting glutamic acid as X.sub.3, and/or having between 7
and 20 amino acid residues, preferably between 10 and 13 amino acid
residues and more preferably between 10 and 11 amino acid
residues.
[0095] It was further described that a conjugate of the peptide
inhibitor described above and a hydrophobic moiety, such as a fatty
acid, attached at the N-terminus of the polypeptide, exerts higher
inhibition of GSK-3 activity (see, for example, WO
2004/052404).
[0096] These peptides were defined as substrate competitive
inhibitors.
[0097] As is well known in the art, substrate competitive enzyme
inhibitors act by binding to the catalytic domain of an enzyme,
thus reducing the proportion of enzyme molecules that are bound to
the enzyme during the catalytic process.
[0098] While recognizing that the development of substrate
competitive inhibitors depends on a molecular understanding of
substrate recognition of protein kinases, efforts have been made in
order to define the catalytic binding site of GSK-3. Thus, Phe67,
Gln89 and Asn95 within the catalytic binding site of GSK-3.beta.
have been reported to play a role in substrates' binding [see,
Ilouz et al., 2006, supra), and a cavity bordered by loop
89-QDKRFKN-95 (as set forth in SEQ ID NO:2), located in the
vicinity of the GSK-3.beta. catalytic core, has been identified as
a promiscuous substrate binding subsite.
[0099] The present inventors have now further explored the role of
the 89-95 loop in GSK-3.beta. substrate binding. To this end, each
of the amino acid residues within this segment was individually
mutated to alanine (see, FIG. 1A). The generated mutants are
denoted herein D90A, K91A, R92A, F93A, K94A, and are represented as
comprising at positions 89-95 an amino acid sequence as set forth
in SEQ ID NOS:6-10, respectively. These mutants were transiently
expressed in HEK-293 cells. These mutants were considerably
expressed, and, similarly to the wild-type (WT) GSK-3.beta., were
phosphorylated at Tyr216 (see, FIG. 1B, lower panel), indicating
that their catalytic activity was not impaired by the mutation
(since phosphorylation at Tyr216 is indicative of an
auto-phosphorylation process).
[0100] The generated GSK-3.beta. mutants were tested in in vitro
kinase assays with known GSK-3 substrates. The mutation at Phe93
was found to exhibit the most pronounced effect for all of tested
substrates, reducing the kinase ability to phosphorylate the
substrate by more than 50% (see, Table 1 hereinbelow and FIG. 1C),
indicating that this position is important for substrate binding,
as previously found for Phe67, Gln89 and Asn95. Phe 93 is located
at the center of the 89-95 loop, it is highly exposed (81% solvent
accessibility) and it faces the substrate binding subsite,
facilitating contacts with a variety of residues. Further studies
substantiated the findings that Phe93 interacts with GSK-3
substrates in cellular conditions (see, FIGS. 1D-1G).
[0101] The role of Phe93 and other amino acids within the 89-95
loop was tested also by determining the inhibitory activity of the
previously described substrate competitive inhibitors L803
(KEAPPAPPQS(p)P; see, SEQ ID NO:4) and its cell permeable variant
L803-mts (see, SEQ ID NO:5). The results indicated that both
L803-mts and L803 did not inhibit the F93A mutated enzyme (see,
FIGS. 3B and 3C), yet inhibited all other mutants, thus further
substantiating the role of Phe93 as a most important binding
position, and the role of hydrophobic interactions also within the
89-95 look as promoting inhibition of GSK-3.
[0102] These findings have led the present inventors to design
novel and improved substrate competitive inhibitors, which exhibit
enhanced interaction with the catalytic binding subsite of GSK-3,
and thus enhanced inhibition activity. Exemplary such novel
peptidic substrate competitive GSK-3 inhibitors were designed after
the recognition motif of HSF, as previously described for, for
example, L803, yet their hydrophobic nature was manipulated by
replacing hydrophilic polar amino acids by hydrophobic amino acids
residues such as alanine and proline. Various substitutions within
the amino acid sequence of L803, as an exemplary substrate
competitive inhibitor, have demonstrated a role for a hydrophobic
amino acid residue at the first position upstream the
phosphorylated serine or threonine residue, leading to a new
generation of substrate competitive inhibitors of GSK-3, which
exhibit improved activity.
[0103] Thus, the studies presented herein identified a role of
Phe93, as well as of other amino acids within the 89-95 loop of a
GSK-3 enzyme, in interacting with GSK-3 substrates and hence with
GSK-3 substrate competitive inhibitors, thereby indicating that a
putative substrate competitive inhibitor should exhibit an
interaction with the Phe93 residue, or with an equivalent amino
acid thereof, in a GSK-3 enzyme.
[0104] As used herein throughout, "GSK-3 enzyme", which is also
referred to herein simply as GSK-3, describes a polypeptide having
an amino acid sequence of a known GSK-3 family member (e.g.,
GSK-3.alpha. or GSK-3.beta.). Unless otherwise indicated, this term
refers to a wild-type GSK-3 enzyme. A GSK-3 enzyme is identified by
the EC number EC 2.7.11.26. While the amino acid of GSK-3 is highly
conserved, a wild-type GSK-3 can be GSK-3 of a mammal (e.g., human)
or of any other organism, including microorganisms. An amino acid
sequence of an exemplary GSK-3, human GSK-3.beta., is set forth in
SEQ ID NO:1. A GSK-3 enzyme as used herein is homologous to SEQ ID
NO:1 by at least 50%, at least 60%, at least 70%, at least 80%, at
least 90% or can be 100 5 homologous.
[0105] By "wild-type" it is meant that the typical form of the
enzyme as it occurs in nature, e.g., in an organism. A wild-type
GSK-3 enzyme encompasses both an enzyme isolated from an organism,
a chemically synthesized enzyme and a recombinantly prepared
enzyme.
[0106] According to an aspect of some embodiments of the present
invention there is provided a GSK-3 substrate competitive inhibitor
comprising at least one moiety that is capable of interacting with
at least one amino acid within the catalytic binding site of a
wild-type GSK-3 enzyme, said at least one amino acid comprising a
phenylalanine residue or an equivalent thereof at position 93 of
said wild-type GSK-3 enzyme.
[0107] As used herein throughout, the term "position" is equivalent
to the term "coordinate" with respect to an amino acid
sequence.
[0108] As used herein, an equivalent amino acid refers to an amino
acid which is homologous (i.e., corresponding in position in either
primary or tertiary structure) and/or analogous to a specific
residue or portion thereof in a given GSK-3 sequence.
[0109] An equivalent amino acid in the context of the Phe93
disclosed herein thus encompasses an analogous aromatic or
otherwise hydrophobic amino acid residue at position 93 of a GSK-3
catalytic binding, as well as a phenylalanine residue or an
analogous amino acid residue thereof which is located at a position
that corresponds, either in primary or tertiary structure, to
position 93 of, for example, GSK-3.beta. enzyme as set forth herein
in SEQ ID NO:1.
[0110] By analogous it is meant, for example, a natural amino acid
that resembles in chemical nature to the amino acid residue (e.g.,
Phe and Tyr are analogous; Asn and Gln are analogous; Leu and Ile
are analogous), or a non-natural amino acid that resembles in
chemical nature to the amino acid residue.
[0111] It has been shown most species exhibit a GSK-3 sequence with
a conserved amino acid sequence of the 89-95 loop, and thus feature
Phe at position 93, while some unicellular species exhibit a GSK-3
sequence in which a Tyr residue or an Ile residue are at position
93. This indicates a general role for hydrophobic interactions with
position 93 of a GSK-3, in almost all species where GSK-3 is
expressed.
[0112] By "moiety" it is meant a chemical group, either per se or
which forms a part of e.g., a chemical compound, an amino acid,
peptide or polypeptide.
[0113] By "interacting" it is meant a chemical interaction as a
result of, for example, hydrophobic interactions, including
aromatic interactions, electrostatic interactions, Van der Waals
interactions and hydrogen bonding.
[0114] Since phenylalanine comprises a hydrophobic aromatic side
chain (phenyl), in some embodiments, the interaction of the
inhibitor with the bonding site of GSK-3 comprises hydrophobic
interactions.
[0115] As used herein, the expressions "phenylalanine at position
93", F93, Phe93, Phe 93, are all used interchangeably to denote the
type of the 93.sup.rd amino acid residue within an amino acid
sequence of GSK-3, when numbered downstream of its N-terminus, or
of an equivalent amino acid thereof, as defined herein.
[0116] Thus, in some embodiments, the moiety that is capable of
interacting with Phe93 in GSK-3, is capable of exhibiting
hydrophobic interactions with the aromatic side chain of Phe93.
[0117] Representative examples of such moieties include, but are
not limited to, hydrocarbons, including alkyls, preferably of 2 or
more carbon atoms, cycloalkyls, aryls, each can optionally be
substituted, heteroalicyclic moieties, and heteroaryl moieties, as
these are defined herein.
[0118] In some embodiments, the moiety is a rigid moiety, namely,
is characterized by a low number of free rotations. Exemplary rigid
moieties include, but are not limited to, cyclic moieties, such as
cycloalkyl, heteroalicyclic, aryl or heteroaryl, with aromatic
cyclic moieties, aryls and heteroaryls being more rigid then others
and hence preferred.
[0119] Thus, in some embodiments, the moiety is an aryl or
heteroaryl, and the inhibitor is a substance that comprises an aryl
or heteroaryl, as defined herein. Such moieties further account for
possible aromatic interactions with Phe93.
[0120] In some embodiments, the inhibitor comprises two or more
moieties that are capable of interacting with Phe93 and optionally
also with other amino acid residues within the catalytic binding
site of GSK-3.
[0121] Thus, in some embodiments, the inhibitor as described herein
is such that is capable of interacting both with Phe93 of GSK-3 and
with one or more additional amino acid residues within the
catalytic binding site of a GSK-3 enzyme.
[0122] Any of the hitherto identified amino acid residues within
the catalytic binding site of GSK-3 are contemplated, including,
but not limited to, those described in Ilouz et al. (2006, supra),
in Dajani et al. (supra) and in and in WO 2005/000192.
[0123] Thus, in addition to comprising a moiety that is capable of
interacting with Phe93, an inhibitor as disclosed herein further
comprises moieties that are capable of interacting with one or more
of such additional amino acid moieties. It is preferred that the
moiety that is capable of interacting with Phe93 and the one or
more additional moieties that are capable of interacting with other
positions within the GSK-3 catalytic domain would be in a suitable
proximity and orientation so as to allow mutual interactions with
the different subsites within the catalytic binding site.
[0124] In some embodiments, an inhibitor as described herein is
such that is capable of interacting, in addition to the Phe93, with
the phosphate binding pocket of GSK-3, namely, with one or more
Arg86, Arg196 and Lys205.
[0125] In some embodiments, an inhibitor as described herein is
such that is capable of interacting, in addition to Phe93, with a
hydrophobic patch that is defined by Val214, I216 and Y216.
[0126] In some embodiments, the inhibitor as described herein is
such that is capable of interacting both with Phe93 of GSK-3 and
with one or more of the additional amino acids Phe67, Gln89, Asp90,
Lys91, Arg92, Lys94 and Asp95 in the GSK-3 enzyme, or with one or
more of Phe67, Gln89, Asp90, Arg92, Lys 94 and Asp95 in the GSK-3
enzyme, or with one or more of Phe67, Gln89 and Asn95.
[0127] Thus, in some embodiments, a GSK-3 inhibitor as described
herein comprises one or more moieties that are capable of
interacting with Phe93 (e.g., hydrophobic moieties), and one or
more moieties that are capable of interacting with Phe67 (e.g.,
hydrophobic moieties). In these embodiments, the hydrophobic
moieties are preferably spaced within the inhibitor in a
configuration (proximity and orientation) that allows interactions
with both amino acids.
[0128] In some embodiments, a GSK-3 inhibitor as described herein
comprises one or more moieties that are capable of interacting with
Phe93 (e.g., hydrophobic moieties), and one or more moieties that
are capable of interacting with Gln89 (e.g., via hydrogen bonding
with its amide). In these embodiments, the moieties are preferably
spaced within the inhibitor in a configuration (proximity and
orientation) that allows interactions with both amino acids.
[0129] In some embodiments, a GSK-3 inhibitor as described herein
comprises one or more moieties that are capable of interacting with
Phe93 (e.g., hydrophobic moieties), and one or more moieties that
are capable of interacting with Asn95 (e.g., via hydrogen bonding
with its amide). In these embodiments, the moieties are preferably
spaced within the inhibitor in a configuration (proximity and
orientation) that allows interactions with both amino acids.
[0130] In some embodiments, a GSK-3 inhibitor as described herein
comprises one or more moieties that are capable of interacting with
Phe93 (e.g., hydrophobic moieties), and one or more moieties that
are capable of interacting with Asn95 and Gln89 (e.g., via hydrogen
bonding with their amide). In these embodiments, the moieties are
preferably spaced within the inhibitor in a configuration
(proximity and orientation) that allows interactions with these
amino acids.
[0131] In some embodiments, a GSK-3 inhibitor as described herein
comprises one or more moieties that are capable of interacting with
Phe93 (e.g., hydrophobic moieties), and one or more moieties that
are capable of interacting with one or both Gln89 and Asn95 (e.g.,
via hydrogen bonding with its amide) and with Phe67 (via
hydrophobic interactions). In these embodiments, the moieties are
preferably spaced within the inhibitor in a configuration
(proximity and orientation) that allows interactions with these
amino acids.
[0132] In some embodiments, a GSK-3 inhibitor as described herein
comprises one or more moieties that are capable of interacting with
Phe93 (e.g., hydrophobic moieties), and one or more moieties that
are capable of interacting with one or more of Arg86, Arg196 and
Lys205 (e.g., via hydrogen bonding with the amine). In these
embodiments, the moieties are preferably spaced within the
inhibitor in a configuration (proximity and orientation) that
allows interactions with these amino acids.
[0133] In some embodiments, a GSK-3 inhibitor as described herein
comprises one or more moieties that are capable of interacting with
Phe93 (e.g., hydrophobic moieties), and one or more moieties that
are capable of interacting with one or more of the amino acids of a
hydrophobic patch within the GSK-3 catalytic binding site as
described herein (e.g., via hydrophobic interactions). In these
embodiments, the moieties are preferably spaced within the
inhibitor in a configuration (proximity and orientation) that
allows interactions with these amino acids.
[0134] Any combination of moieties that are suitably spaced in a
configuration that allows interactions with any combination of the
amino acids described herein are also contemplated for an inhibitor
as described herein.
[0135] Determining is a substance or a moiety is capable of
interacting with Phe93 or an equivalent amino acid thereof can be
performed by methods known in the art, as is further detailed
hereinbelow. In some embodiments, computational modeling can be
used to evaluate the interaction of a substance with Phe93. In some
embodiments, the activity of a wild-type GSK-3 and the activity of
a corresponding mutated GSK-3, in which Phe93 is substituted by
another amino acid (e.g., alanine), is determined in the presence
of the inhibitor. Reduction of the inhibition activity of the
substance when tested with the mutated GSK-3, compared to its
inhibition activity of the wild-type GSK-3, is indicative of an
interaction of the inhibitor with Phe93.
[0136] The substrate competitive inhibitor disclosed herein can be
a small molecule, namely a non-peptidic organic compound. Exemplary
compounds include compounds possessing one or more of the
hydrophobic moieties as described herein (e.g., aryl or heteroaryl
moieties) and optionally one or more moieties that are capable of
interacting with one or more of the additional amino acid residues
within the catalytic bonding site, as described herein.
[0137] In some embodiments, the substrate competitive inhibitor is
a peptide (or a polypeptide).
[0138] In some embodiments, the peptide comprises one or more of a
hydrophobic amino acid residue, as defined herein, which are
suitably positioned with respect to other functional amino acid
moieties so as to allow interactions with other subsites within the
catalytic binding site of GSK-3 (e.g., the phosphate binding
pocket).
[0139] In some embodiments, the peptide is based on a recognition
motif of a GSK-3 substrate as defined herein and was previously
described (see, for example, WO 01/49709).
[0140] Excluded from the scope of these embodiments of the present
invention are substances, including small molecules and peptides
already reported in the art as acting as GSK-3 inhibitors. These
include, for example, substrate competitive inhibitors of GSK-3
inhibitors as described in Plotkin et al. (2003) J. Pharmacol. Exp.
Ther., 974-980], in Kaidanovich-Beilin & Eldar-Finkelman (2005)
J. Pharmacol. Exp. Ther. 316:17-24; in Rao et al. (2007)
Diabetologia 50, 452-60; Kim et al. (2006) Neuron 52, 981-96; in
Chen et al. (2004) Faseb J 18, 1162-4; in Kaidanovich-Beilin et al.
(2004) Biol. Psychiatry. 55:781-4; in Shapira et al. (2007) Mol.
Cell Neurosci. 34, 571-7]; in Ilouz et al. (2006) J. Biol. Chem.
281, 30621-30]; in U.S. Pat. Nos. 6,780,625 and 7,378,432; in WO
2004/052404, WO 2005/000192; and WO 01/49709; in Liberman, Z. &
Eldar-Finkelman, H. (2005) J. Biol. Chem. 280, 4422-8; in Liberman
et al. (2008) Am. J. Physiol. Endocrinol. Metab. 294, E1169-77; and
in Bertrand et al. (2003) J. Mol. Biol. 333, 393-407.
[0141] As discussed hereinabove, the present inventors have
designed novel peptides, which are based on the a recognition motif
of a GSK-3 substrate, and are further designed to feature defined
characteristics which provide for increased interaction of the
peptide with the catalytic binding site of GSK-3, and particularly
with Phe93 (or an equivalent amino acid residue, as defined
herein).
[0142] Thus, newly designed peptides are disclosed herein. These
peptides are collectively represented by the amino acid sequence I
as follows:
[Yn . . . Y.sub.1]ZX.sub.1X.sub.2X.sub.3S(p)[W.sub.1 . . . Wm]
(I)
[0143] wherein,
[0144] m equals 1 or 2;
[0145] n is an integer from 3 to 7, such that said polypeptide
consists of 10 to 13 amino acid residues;
[0146] S(p) is a phosphorylated serine residue or a phosphorylated
threonine residue;
[0147] Z is any amino acid residue excepting serine residue or
threonine residue;
[0148] X.sub.1, X.sub.2, Y.sub.1-Yn and W.sub.1-Wm are each
independently any amino acid residue; and
[0149] X.sub.3 is a hydrophobic amino acid residue.
[0150] According to some embodiments of the present invention, the
peptides described herein can be considered as sequenced based on a
natural or otherwise identified GSK-3 substrate (e.g., CREB or
HSF-1), while maintaining the identified recognition motif of GSK-3
described hereinabove (see, SEQ ID NO:3), which includes
phosphorylated serine or threonine residue, and while replacing the
serine or threonine that is at the fourth position upstream of the
phosphorylated serine or threonine.
[0151] The phrase "natural GSK-3 substrate" or "known GSK-3
substrate" describes any peptide (or protein) which is known to be
phosphorylated by GSK-3 in a biological system. By "biological
system" it is meant a system of any living species including, for
example, vertebrates, poultry, mammals, human beings and
microorganisms, including unicellular organisms. Representative
examples of natural GSK-3 substrates include, but are not limited
to, HSF-1, pIRS-1, p9CREB, pGS-1, phosphorylated peptides derived
from the insulin receptor substrate-1 (IRS-1) [see, for example,
Liberman and Eldar-Finkelman (2005) supra], cAMP responsive element
binding protein (CREB), and glycogen synthase, some of which are
set forth herein as having SEQ ID NOS:18-20.
[0152] It is expected that during the life of a patent maturing
from this application additional relevant GSK-substrates will be
identified and the scope of the term "natural GSK-3 substrate" is
intended to include all such new substrates a priori.
[0153] As discussed hereinabove, in the peptides described herein,
the amino acid residue at the first position upstream of the
phosphorylated serine or threonine (denoted as X.sub.3) is a
hydrophobic amino acid residue.
[0154] Thus, in some embodiments, the peptides described herein can
be considered as sequenced based on a natural or otherwise
identified GSK-3 substrate, while maintaining the identified
recognition motif of GSK-3 described hereinabove (see, SEQ ID
NO:3), which includes phosphorylated serine or threonine residue,
and while replacing the serine or threonine residue that is at the
fourth position upstream of the phosphorylated serine or threonine
by any other amino acid residue and while replacing the amino acid
residue at the first position upstream of the phosphorylated serine
or threonine by a hydrophobic amino acid residue.
[0155] The term "hydrophobic", as used herein with reference to an
amino acid or any other substance or moiety, describes a feature of
the substance that renders its solubility in water lower than its
solubility in hydrophobic organic solvents.
[0156] The term "hydrophobic" thus often translates into values
such as Log P, which describes the partition coefficient of a
substance between an aqueous phase (water) and an oily phase
(1-octanol).
[0157] According to some embodiments of the present invention, a
hydrophobic amino acid has a Log P value that is higher (i.e., less
negative) than -3, or higher than -2.9, or higher than -2.8, or
higher than -2.7, or higher than -2.6, or even higher than
-2.5.
[0158] Exemplary hydrophobic amino acids include, but are not
limited to, glycine, alanine, leucine, isoleucine, valine, proline,
phenylalanine, methionine, cysteine and tryptophan.
[0159] In some embodiments, X.sub.3 is a proline residue or an
alanine residue.
[0160] In some embodiments, X.sub.3 is a proline residue.
[0161] In some embodiments, X.sub.3 is an amino acid that an a
hydrophobic side chain which is rigid, thus ensuring better
interaction (reduced entropy) with the catalytic binding site.
Exemplary such amino acids have a side chain that comprises an aryl
(e.g., tryptophan and phenylalanine) or a heteroaryl (e.g.,
proline).
[0162] X.sub.1 and X.sub.2 in the amino acid sequence of the
peptide described herein can be any amino acid, as described
herein.
[0163] In some embodiments, at least one, or both, of X.sub.1 and
X.sub.2 is a hydrophobic amino acid, as described herein.
[0164] Thus, in some embodiments, each of X.sub.1, X.sub.2 and
X.sub.3 is a hydrophobic amino acid residue, as described herein
(e.g., alanine or proline).
[0165] In some embodiments, X.sub.1 and X.sub.2 are each a proline
residue.
[0166] In some embodiments, each of X.sub.1, X.sub.2 and X.sub.3 is
a proline residue.
[0167] In some embodiments, S(p) is a phosphorylated serine
residue.
[0168] The amino acid denoted Z in the amino acid sequence of the
peptide described herein can be any amino acid, as described
herein.
[0169] In some embodiments, Z is an alanine residue.
[0170] In some embodiments, a peptide as described herein comprises
any one of the following amino acid sequences as the moiety denoted
as ZX.sub.1X.sub.2X.sub.3S(p) in amino acid sequence I, as
non-limiting examples:
[0171] Ala-Pro-Pro-Pro-phosphorylated serine (SEQ ID NO:21)
[0172] Ala-Pro-Pro-Pro-phosphorylated threonine (SEQ ID NO:22)
[0173] Ala-Ala-Pro-Pro-phosphorylated serine (SEQ ID NO:23)
[0174] Ala-Ala-Pro-Pro-phosphorylated threonine (SEQ ID NO:24)
[0175] Ala-Ala-Ala-Pro-phosphorylated serine (SEQ ID NO:25)
[0176] Ala-Ala-Ala-Pro-phosphorylated threonine (SEQ ID NO:26)
[0177] Ala-Pro-Ala-Pro-phosphorylated serine (SEQ ID NO:27)
[0178] Ala-Pro-Ala-Pro-phosphorylated threonine (SEQ ID NO:28)
[0179] Ala-Gly-Pro-Pro-phosphorylated serine (SEQ ID NO:29)
[0180] Ala-Gly-Pro-Pro-phosphorylated threonine (SEQ ID NO:30)
[0181] Ala-Gly-Gly-Pro-phosphorylated serine (SEQ ID NO:31)
[0182] Ala-Gly-Gly-Pro-phosphorylated threonine (SEQ ID NO:32)
[0183] Ala-Pro-Gly-Pro-phosphorylated serine (SEQ ID NO:33)
[0184] Ala-Pro-Gly-Pro-phosphorylated threonine (SEQ ID NO:34)
[0185] Ala-Leu/Ile-Pro-Pro-phosphorylated serine (SEQ ID NO:35)
[0186] Ala-Leu/Ile-Pro-Pro-phosphorylated threonine (SEQ ID
NO:36)
[0187] Ala-Leu/Ile-Leu/Ile-Pro-phosphorylated serine (SEQ ID
NO:37)
[0188] Ala-Leu/Ile-Leu/Ile-Pro-phosphorylated threonine (SEQ ID
NO:38)
[0189] Ala-Pro-Leu/Ile-Pro-phosphorylated serine (SEQ ID NO:39)
[0190] Ala-Pro-Leu/Ile-Pro-phosphorylated threonine (SEQ ID
NO:40)
[0191] Ala-Val-Pro-Pro-phosphorylated serine (SEQ ID NO:41)
[0192] Ala-Val-Pro-Pro-phosphorylated threonine (SEQ ID NO:42)
[0193] Ala-Val-Val-Pro-phosphorylated serine (SEQ ID NO:43)
[0194] Ala-Val-Val-Pro-phosphorylated threonine (SEQ ID NO:44)
[0195] Ala-Pro-Val-Pro-phosphorylated serine (SEQ ID NO:45)
[0196] Ala-Pro-Val-Pro-phosphorylated threonine (SEQ ID NO:46).
[0197] In some embodiments, in any of these moieties, the Pro
residue at the first position upstream the phosphorylated serine or
threonine (X.sub.3) is replaced by any of the other hydrophobic
moieties as described herein (e.g., Phe or Trp).
[0198] It is to be noted that for X.sub.1, X.sub.2 and X.sub.3, any
combination of 3 hydrophobic amino acid residues as defined herein
is contemplated in some embodiments of the present invention, and
that any such combination can be combined with either a
phosphorylated serine residue or a phsophorylated threonine residue
at the position denoted S(p), and with any amino acid residue at
the position denoted Z.
[0199] In some embodiments, the number and nature of amino acid
residues downstream the residue denoted as S(p) and upstream the
residue denoted as Z, is determined by the amino acid sequence of
the GSK-3 substrate after which the peptide is designed.
[0200] In some embodiments, m is 1.
[0201] In some embodiments, W.sub.1 is a proline residue, although
any other amino acid residue at this position, and at position
W.sub.2 (if present, when m=2) is also contemplated.
[0202] In some embodiments, n is 5, such that the peptide comprises
an amino acid sequence as described herein, in which upstream to Z
there are amino acid residues denoted as Y.sub.1-Y.sub.5.
[0203] In some embodiments, when the peptide is designed after the
substrate HSF-1, Y.sub.1-Y.sub.5 has the amino acid sequence
Lys-Glu-Ala-Pro-Pro, as set forth in any of SEQ ID NOS:11-16.
However, any other sequence of amino acid residues can be included
within the amino acid residues upstream to Z.
[0204] In other embodiments, Y.sub.3--Y.sub.5 are each a
hydrophobic amino acid residue (as defined herein, e.g., proline
and/or alanine), and at least one of Y.sub.1 and Y.sub.2 is a
hydrophobic amino acid residue (as defined herein, e.g., proline or
alanine).
[0205] Exemplary peptides are those having the following amino acid
sequences:
[0206] Lys-Glu-Ala-Pro-Pro-Ala-Pro-Pro-Pro-phosphorylated Ser-Pro
(PQ9P; SEQ ID NO:16);
[0207] Lys-Glu-Ala-Pro-Pro-Ala-Pro-Pro-Ala-phosphorylated Ser-Pro
(PQ9A; SEQ ID NO:13);
[0208] Ala-Glu-Ala-Pro-Pro-Ala-Pro-Pro-Gln-phosphorylated Ser-Pro
(PK1A; SEQ ID NO:11);
[0209] Lys-Ala-Ala-Pro-Pro-Ala-Pro-Pro-Gln-phosphorylated Ser-Pro
(PE2A; SEQ ID NO:12).
[0210] In some embodiments, the polypeptide has the amino acid
sequence: Lys-Glu-Ala-Pro-Pro-Ala-Pro-Pro-Pro-phosphorylated
Ser-Pro (PQ9P; SEQ ID NO:16).
[0211] In addition to the inclusion of hydrophobic amino acid
residues within the amino acid sequence of the peptide described
herein, as presented herein, any of the peptides described herein
can further comprise a hydrophobic moiety covalently attached
thereto.
[0212] As used herein the phrase "hydrophobic moiety" refers to any
substance that is characterized by hydrophobicity, namely, its
solubility in water is much lower than its solubility in
hydrophobic organic solvents, as defined herein.
[0213] In some embodiments, any hydrophobic moiety that is
structurally suitable for interacting with a hydrophobic patch
within a GSK-3 dimer, can be attached to the polypeptide described
above.
[0214] The hydrophobic patch has been previously described by
Dajani et al. (2001, supra). The crystallization data of Dajani et
al. showed that GSK-3 is crystallized as a dimer, suggesting that
this dimerization has biological relevance. The catalytic region
(residues 216-220) of one monomer (a) appears to interact with the
N-terminus of an .alpha.-helix (residues 262-273) of the other
monomer (b). This interaction of the two monomers (a) and (b) forms
a hydrophobic patch in monomer (b).
[0215] Alternatively, or in addition, the hydrophobic moiety is
selected such that it enhances cell permeability of the peptide.
Enhanced cell permeability can be determined by any method known in
the art, for example, by determining a cellular uptake in in vitro
studies.
[0216] Representative examples of hydrophobic substances from which
the hydrophobic moiety of the present invention can be derived
include, without limitation, substituted and unsubstituted,
saturated and unsaturated hydrocarbons, where the hydrocarbon can
be an aliphatic, an alicyclic or an aromatic compound and
preferably includes at least 4 carbon atoms, more preferably at
least 8 carbon atoms, more preferably at least 10 carbon atoms. In
some embodiments, the hydrocarbon bears a functional group which
enables its attachment to an amino acid residue. Representative
examples of such a functional group include, without limitation, a
free carboxylic acid (C(.dbd.O)OH), a free amino group (NH.sub.2),
an ester group (C(.dbd.O)OR, where R is alkyl, cycloalkyl or aryl),
an acyl halide group (C(.dbd.O)A, where A is fluoride, chloride,
bromide or iodide), a halide (fluoride, chloride, bromide or
iodide), a hydroxyl group (OH), a thiol group (SH), a nitrile group
(C.ident.N), a free C-carbamic group (NR''--C(.dbd.O)--OR', where
each of R' and R'' is independently hydrogen, alkyl, cycloalkyl or
aryl), a free N-carbamic group (OC(.dbd.O)--NR'--, where R' is as
defined above), a thionyl group (S(.dbd.O).sub.2A, where A is
halide as defined above) and the like.
[0217] In some embodiments, the hydrophobic moiety comprises one or
more fatty acid(s).
[0218] Representative examples of fatty acids that are usable in
the context of the present invention include, without limitation,
saturated or unsaturated fatty acids that have more than 10 carbon
atoms, preferably between 12 and 24 carbon atoms, such as, but not
limited to, myristic acid, lauric acid, palmitic acid, stearic
acid, oleic acid, linoleic acid, linolenic acid, arachidonic etc.,
with myristic acid being presently the most preferred.
[0219] The hydrophobic moiety according to some embodiments of the
present invention can be a fatty acid, or derived from any other
hydrophobic substance as described above, per se, such that the
fatty acid, or any other hydrophobic substance, is covalently
attached directly to an amino acid residue of the peptide (via, for
example, en ester bond or an amide bond). Alternatively, the
hydrophobic moiety can be an amino acid residue that is modified to
include a fatty acid, or any other hydrophobic substance as
described hereinabove, such that this modified amino acid residue
is attached to the peptide via a peptide bond or a substituted
peptide bond, as is described herein. Further alternatively, the
hydrophobic moiety can be a short peptide in which one or more
amino acid residues are modified to include a fatty acid or any
other hydrophobic substance as described herein. Such a peptide
preferably includes between 2 and 15 amino acid residues and is
attached to the peptide via a peptide bond or a substituted peptide
bond, as is described herein.
[0220] As an alternative to, or in combination with the hydrophobic
moiety described above, the hydrophobic moiety, according to the
present invention, can comprise a hydrophobic peptide sequence. The
hydrophobic peptide sequence, according to the present invention,
preferably includes between 2 and 15 amino acid residues, more
preferably between 2 and 10 amino acid residues, more preferably
between 2 and 5 amino acid residues, in which at least five
consecutive amino acid residues are hydrophobic amino acid
residues.
[0221] Alternatively, the hydrophobic amino acid residue can
include any other amino acid residue, which has been modified by
incorporation of a hydrophobic moiety thereto.
[0222] The hydrophobic moiety or moieties of the present invention
are preferably attached to one or more termini of the peptide,
namely the N-terminus and/or the C-terminus of the polypeptide. In
some embodiments, the hydrophobic moiety is attached, directly or
indirectly, as described herein, to the N-terminus of the
polypeptide.
[0223] An exemplary peptide has the amino acid sequence
Myristic-Gly-Lys-Glu-Ala-Pro-Pro-Ala-Pro-Pro-Pro-phosphorylated
Ser-Pro (PQ9P; SEQ ID NO:17).
[0224] Additional exemplary peptides are those represented by any
of SEQ ID NOS:11-13, and/or those comprising any of SEQ ID
NOS:21-46, as described herein, which have a hydrophobic moiety
attached thereto, as described herein. Any combination of such
peptides and a hydrophobic moiety as described herein (e.g., a
fatty acid as described herein and/or an amino acid substituted by
a fatty acid as described herein and/or a hydrophobic amino acid
sequence as described herein) is contemplated.
[0225] Further according to embodiments of the present invention,
there is provided a process of preparing the peptides described
herein.
[0226] In one embodiment, the peptide of the present invention is
prepared by a chemical synthesis, using well known chemical
procedures, such as solution or solid-phase peptide synthesis, or
semi-synthesis in solution. The peptide can be chemically
synthesized, for example, by the solid phase peptide synthesis of
Merrifield et al (1964). Alternatively, a peptide can be
synthesized using standard solution methods (see, for example,
Bodanszky, 1984). Newly synthesized peptides can be purified, for
example, by high performance liquid chromatography (HPLC), and can
be characterized using, for example, mass spectrometry or amino
acid sequence analysis.
[0227] Alternatively, the peptides of the invention can be provided
recombinantly. Systems for cloning and expressing the peptide
include various microorganisms and cells that are well known in
recombinant technology. These include, for example, various strains
of E. coli, Bacillus, Streptomyces, and Saccharomyces, as well as
mammalian, yeast and insect cells. The peptide can be produced as a
peptide or fusion protein (e.g., tagged peptide). Suitable vectors
for producing the peptide are known and available from private and
public laboratories and depositories and from commercial vendors.
See Sambrook et al, (1989). Recipient cells capable of expressing
the gene product are then transfected. The transfected recipient
cells are cultured under conditions that permit expression of the
recombinant gene products, which are recovered from the culture.
Host mammalian cells, such as Chinese Hamster ovary cells (CHO) or
COS-1 cells, can be used. These hosts can be used in connection
with poxvirus vectors, such as vaccinia or swinepox. Suitable
non-pathogenic viruses that can be engineered to carry the
synthetic gene into the cells of the host include poxviruses, such
as vaccinia, adenovirus, retroviruses and the like. A number of
such non-pathogenic viruses are commonly used for human gene
therapy, and as carrier for other vaccine agents, and are known and
selectable by one of skill in the art. The selection of other
suitable host cells and methods for transformation, culture,
amplification, screening and product production and purification
can be performed by one of skill in the art by reference to known
techniques (see, e.g., Gething et al, 1981).
[0228] Once the peptide is provided, a hydrophobic moiety or
moieties can be conjugated thereto, if desired, by commonly used
techniques. For example, in cases where the hydrophobic moiety is a
fatty acid, techniques for adding a fatty acid (e.g., myristic
acid) to an amino acid residue within the peptide sequence are
used. Alternatively, an amino acid residue is modified to include a
hydrophobic moiety such as fatty acid and is thereafter attached to
the peptide by known chemical procedures, as is described
hereinabove.
[0229] In cases where the hydrophobic moiety comprises a
hydrophobic peptide sequence, the hydrophobic peptide can be
prepared using the methods described hereinabove and thereafter be
conjugated to the polypeptide. Alternatively, the conjugate can be
prepared recombinantly, using systems, as described hereinabove,
for cloning and expressing a fused polypeptide that comprises the
peptide as described herein and such a hydrophobic peptide
sequence.
[0230] As is demonstrated in the Examples section that follows,
exemplary peptides according to some embodiments of the present
invention exhibit high inhibitory effect toward GSK-3.
[0231] As is discussed hereinabove, these peptides are
characterized by specificity towards GSK-3, a specificity which is
derived from the unique recognition motif of GSK-3, which, unlike
other kinases, includes a phosphorylated serine or threonine
residue, and the fact that the sequence of the peptide portion
thereof is based on this recognition motif.
[0232] The additional manipulation made to the GSK-3 recognition
motif while designing the peptides disclosed herein render these
peptides efficient substrate competitive inhibitors of GSK-3, and
thus more specific as compared with other protein kinase inhibitors
that are typically ATP competitive compounds and thus
non-specific.
[0233] Thus, the high inhibitory activity of the peptides disclosed
herein is derived from both, the replacement of the phosphorylated
residue at the Z position by a non-phosphorylated residue, which
renders the enzyme inactive in phosphorylation, and the
incorporation of a hydrophobic amino acid residue at the indicated
position within the recognition motif, which provides for enhanced
interaction with a subunit of the enzyme's catalytic binding site,
as discussed herein.
[0234] Hence, according to another aspect of some embodiments of
the present invention, there is provided a method of inhibiting an
activity of GSK-3, which is effected by contacting cells expressing
GSK-3 with an effective amount of any of the peptides described
herein (e.g., represented by amino acid sequence I), or by any of
the GSK-3 substrate competitive inhibitors as described herein
(which are capable of interacting with Phe93 or an equivalent
thereof).
[0235] As used herein, the term "effective amount" is the amount
determined by such considerations as are known in the art, which is
sufficient to reduce the activity of GSK-3 by at least 5%, at least
10%, at least 20%, at least 50% and even at least 80%, 90% or by
100%. Typical assays for measuring kinase activity can be used for
determining the inhibitory activity of the peptides as described
herein.
[0236] As is demonstrated in the Examples section that follows, a
representative example of a peptide according to some embodiments
of the present invention strongly inhibits GSK-3, with an IC.sub.50
value of less than 50 .mu.M, and even less than 1 .mu.M, as
measured by in vitro kinase assay.
[0237] Hence, the effective amount of a peptide as described herein
can range from about 0.1 micromolar to about 100 micromolar, or
from about 0.1 micromolar and about 50 micromolar, or from about
0.1 micromolar to about 20 micromolar, or from about 1 micromolar
to about 20 micromolar, including any intermediate value between
the indicated ranges.
[0238] As used herein throughout the term "about" refers to
.+-.10%.
[0239] As is further demonstrated in the Examples section that
follows, the inhibition activity of the peptides described herein
was tested in both in vitro and in vivo assays. Thus, the method
according to this aspect of the present invention can be effected
by contacting the cells with the described peptides in vitro, ex
vivo and in vivo.
[0240] Cells expressing GSK-3 can be derived from any biological
sample, including, but not limited to, cell cultures or extracts
thereof, enzyme preparations suitable for in vitro assays, biopsied
material obtained from a mammal or extracts thereof, and samples of
blood, saliva, urine, feces, semen, tears, spinal fluid, and any
other fluids or extracts thereof.
[0241] In some embodiments, the method according to these
embodiments, utilizes the peptides as described herein as active
agents in biological assays, and in particular, as GSK-3 (substrate
competitive) inhibitors in such assays.
[0242] As the peptides described herein do not include the required
phosphorylated residue (at the Z position), GSK-3, while being
bound thereto, is rendered inactive in phosphorylation reactions.
Thus, the method according to these embodiments of the present
invention preferably pertains to inhibition of the phosphorylation
and/or autophosphorylation activity of GSK-3. In some embodiments,
the activity is phosphorylation activity.
[0243] The method according to these embodiments of the present
invention can be further effected by contacting the cells with an
additional active ingredient that is capable of altering an
activity of GSK-3, as is detailed hereinbelow.
[0244] The inhibition of GSK-3 activity is a way to increase
insulin activity in vivo. High activity of GSK-3 impairs insulin
action in intact cells. This impairment results from the
phosphorylation of insulin receptor substrate-1 (IRS-1) serine
residues by GSK-3. Studies performed in patients with type II
diabetes (non-insulin dependent diabetes mellitus, NIDDM) show that
glycogen synthase activity is markedly decreased in these patients,
and that decreased activation of protein kinase B (PKB), an
upstream regulator of GSK-3, by insulin is also detected. Mice
susceptible to high fat diet-induced diabetes and obesity have
significantly increased GSK-3 activity in epididymal fat tissue.
Increased GSK-3 activity expressed in cells resulted in suppression
of glycogen synthase activity.
[0245] Inhibition of GSK-3 activity therefore provides a useful
method for increasing insulin activity in insulin-dependent
conditions. For example, treatment with the peptides as described
herein can result in improved glucose uptake and/or glucose
tolerance.
[0246] Thus, according to another aspect of the present invention
there is provided a method of potentiating insulin signaling, which
is effected by contacting insulin responsive cells with an
effective amount, as is defined hereinabove, of the peptide as
described herein.
[0247] Contacting can be effected in vitro, as described herein,
for example, by contacting a biological sample as described herein
with one or more of the peptides described herein, or ex vivo, or
in vivo, by administering a peptide as described herein to a
patient in need thereof.
[0248] As used herein, the phrase "potentiating insulin signaling"
includes an increase in the phosphorylation of insulin receptor
downstream components and an increase in the rate of glucose uptake
as compared with glucose uptake in untreated subjects or cells.
[0249] Potentiation of insulin signaling, in vivo, resulting from
administration of the peptides as described herein, can be
monitored as a clinical endpoint. In principle, the easiest way to
look at insulin potentiation in a patient is to perform the glucose
tolerance test. After fasting, glucose is given to a patient and
the rate of the disappearance of glucose from blood circulation
(namely glucose uptake by cells) is measured by assays well known
in the art. Slow rate (as compared to healthy subject) of glucose
clearance will indicate insulin resistance. The administration of a
GSK-3 inhibitor such as the peptides described herein to an
insulin-resistant patient increases the rate of glucose uptake as
compared with a non-treated patient. The peptide may be
administered to the patient for a longer period of time, and the
levels of insulin, glucose, and leptin in blood circulation (which
are usually high) may be determined. Decrease in glucose levels
will indicate that the peptide potentiated insulin action. A
decrease in insulin and leptin levels alone may not necessarily
indicate potentiation of insulin action, but rather will indicate
improvement of the disease condition by other mechanisms.
[0250] By inhibiting GSK-3 activity and/or potentiating insulin
signaling, the peptides described herein may be effectively
utilized for treating any biological condition that is associated
with GSK-3.
[0251] Hence, according to another aspect of some embodiments of
the present invention, there is provided a method of treating a
biological condition associated with GSK-3 activity. The method,
according to this aspect of the present invention, is effected by
administering to a subject in need thereof a therapeutically
effective amount of the peptide as described herein.
[0252] The phrase "biological condition associated with GSK-3
activity" as used herein includes any biological or medical
condition or disorder in which effective GSK-3 activity is
identified, whether at normal or abnormal levels. The condition or
disorder may be caused by the GSK-3 activity or may simply be
characterized by GSK-3 activity. That the condition is associated
with GSK-3 activity means that some aspects of the condition can be
traced to the GSK-3 activity. Such a biological condition can also
be regarded as a biological or medical condition mediated by
GSK-3.
[0253] Herein, the term "treating" includes abrogating,
substantially inhibiting, slowing or reversing the progression of a
condition or disorder, substantially ameliorating clinical symptoms
of a condition or disorder or substantially preventing the
appearance of clinical symptoms of a condition or disorder. These
effects may be manifested, for non-limiting examples, by a decrease
in the rate of glucose uptake with respect to type II diabetes or
by halting neuronal cell death with respect to neurodegenerative
disorders, as is detailed hereinbelow.
[0254] The term "administering" as used herein describes a method
for bringing a peptide as described herein and cells affected by
the condition or disorder together in such a manner that the
peptide can affect the GSK-3 activity in these cells. The peptides
described herein can be administered via any route that is
medically acceptable. The route of administration can depend on the
disease, condition, organ or injury being treated. Possible
administration routes include injections, by parenteral routes,
such as intravascular, intravenous, intra-arterial, subcutaneous,
intramuscular, intratumor, intraperitoneal, intraventricular,
intraepidural, intracerebroventricular, intranasal or others, as
well as via oral, nasal, ophthalmic, rectal or topical routes of
administration, or by inhalation. Sustained release administration
is also encompassed herein, by means such as, for example, depot
injections or erodible implants, or by sustained release oral
formulations (e.g., solid oral formulations). Administration can
also be intra-articularly, intrarectally, intraperitoneally,
intramuscularly, subcutaneously, or by aerosol inhalant. Where
treatment is systemic, the peptide can be administered orally,
nasally or parenterally, such as intravenously, intramuscularly,
subcutaneously, intraorbitally, intracapsularly, intraperitoneally
or intracisternally, as long as provided in a composition suitable
for effecting the introduction of the peptide into target cells, as
is detailed hereinbelow.
[0255] In some embodiments, administration is effected nasally,
namely via a nasal route of administration. A nasal administration
can be effected either by intranasal injection or by means of a
spray or liquid formulation that is administered nasally.
[0256] The phrase "therapeutically effective amount", as used
herein, describes an amount administered to an individual, which is
sufficient to abrogate, substantially inhibit, slow or reverse the
progression of a condition associated with GSK-3 activity, to
substantially ameliorate clinical symptoms of a such a condition or
substantially prevent the appearance of clinical symptoms of such a
condition. The GSK-3 activity can be a GSK-3 kinase activity. The
inhibitory amount may be determined directly by measuring the
inhibition of a GSK-3 activity, or, for example, where the desired
effect is an effect on an activity downstream of GSK-3 activity in
a pathway that includes GSK-3, the inhibition may be measured by
measuring a downstream effect. Thus, for example where inhibition
of GSK-3 results in the arrest of phosphorylation of glycogen
synthase, the effects of the peptide may include effects on an
insulin-dependent or insulin-related pathway, and the peptide may
be administered to the point where glucose uptake is increased to
optimal levels. Also, where the inhibition of GSK-3 results in the
absence of phosphorylation of a protein that is required for
further biological activity, for example, the tau protein, then the
peptide may be administered until polymerization of phosphorylated
tau protein is substantially arrested. Level of hippocampous
.beta.-catenin are also indicative for an effect on GSK-3 activity.
Therefore, the inhibition of GSK-3 activity will depend in part on
the nature of the inhibited pathway or process that involves GSK-3
activity, and on the effects that inhibition of GSK-3 activity has
in a given biological context.
[0257] The amount of the peptide that will constitute an inhibitory
amount will vary depending on such parameters as the peptide and
its potency, the half-life of the peptide in the body, the rate of
progression of the disease or biological condition being treated,
the responsiveness of the condition to the dose of treatment or
pattern of administration, the formulation, the attending
physician's assessment of the medical situation, and other relevant
factors, and in general the health of the patient, and other
considerations such as prior administration of other therapeutics,
or co-administration of any therapeutic that will have an effect on
the inhibitory activity of the peptide or that will have an effect
on GSK-3 activity, or a pathway mediated by GSK-3 activity.
[0258] Although it is expected that the inhibitory amount will fall
in a relatively broad range that can be determined through routine
trials, an exemplary therapeutically effective amount according to
the present invention is selected so as to achieve, at the treated
site, an amount of the peptide that ranges between about 10 nmol
and about 1000 nmol, or between about 10 nmol and about 500 nmol,
or between about 100 nmol and about 400 nmol.
[0259] As is discussed in detail hereinabove, GSK-3 is involved in
various biological pathways and hence, the method according to this
aspect of the present invention can be used in the treatment of a
variety of biological conditions, as is detailed hereinunder.
[0260] GSK-3 is involved in the insulin signaling pathway and
therefore, in one example, the method according this aspect of the
present invention can be used to treat any insulin-dependent
condition.
[0261] By "insulin-dependent condition" it is meant any condition
that is mediated by insulin and which is manifested or caused by
reduced level of insulin or impaired insulin potentiation pathway.
Exemplary such conditions include, but are not limited to,
conditions that involve glucose intolerance and impaired glucose
uptake, such as diabetes, including, for example, insulin-dependent
diabetes and juvenile diabetes.
[0262] As GSK-3 inhibitors are known to inhibit differentiation of
pre-adipocytes into adipocytes, in another example, the method of
this aspect of the present invention can be used to treat
obesity.
[0263] In yet another example, the method according to this aspect
of the present invention can be used to treat diabetes including
non-insulin dependent diabetes mellitus.
[0264] Diabetes mellitus is a heterogeneous primary disorder of
carbohydrate metabolism with multiple etiologic factors that
generally involve insulin deficiency or insulin resistance or both.
Type I, juvenile onset, insulin-dependent diabetes mellitus, is
present in patients with little or no endogenous insulin secretory
capacity. These patients develop extreme hyperglycemia and are
entirely dependent on exogenous insulin therapy for immediate
survival. Type II, or adult onset, or non-insulin-dependent
diabetes mellitus, occurs in patients who retain some endogenous
insulin secretory capacity, but the great majority of them are both
insulin deficient and insulin resistant. Approximately 95% of all
diabetic patients in the United States have non-insulin dependent,
Type II diabetes mellitus (NIDDM), and, therefore, this is the form
of diabetes that accounts for the great majority of medical
problems. Insulin resistance is an underlying characteristic
feature of NIDDM and this metabolic defect leads to the diabetic
syndrome. Insulin resistance can be due to insufficient insulin
receptor expression, reduced insulin-binding affinity, or any
abnormality at any step along the insulin signaling pathway (see
U.S. Pat. No. 5,861,266).
[0265] The peptides described herein can be used to treat type II
diabetes in a patient with type II diabetes as follows: a
therapeutically effective amount of the peptide is administered to
the patient, and clinical markers, e.g., blood sugar level, are
monitored. The peptide can further be used to prevent type II
diabetes in a subject as follows: a prophylactically effective
amount of the peptide is administered to the patient, and a
clinical marker, for example IRS-1 phosphorylation, is
monitored.
[0266] Treatment of diabetes is determined by standard medical
methods. A goal of diabetes treatment is to bring sugar levels down
to as close to normal as is safely possible. Commonly set goals are
80-120 milligrams per deciliter (mg/dl) before meals and 100-140
mg/dl at bedtime. A particular physician may set different targets
for the patent, depending on other factors, such as how often the
patient has low blood sugar reactions. Useful medical tests include
tests on the patient's blood and urine to determine blood sugar
level, tests for glycated hemoglobin level (HbA.sub.1c; a measure
of average blood glucose levels over the past 2-3 months, normal
range being 4-6%), tests for cholesterol and fat levels, and tests
for urine protein level. Such tests are standard tests known to
those of skill in the art (see, for example, American Diabetes
Association, 1998). A successful treatment program can also be
determined by having fewer patients in the program with diabetic
eye disease, kidney disease, or nerve disease.
[0267] Hence, in one particular embodiment of the method according
to this aspect of the present invention, there is provided a method
of treating non-insulin dependent diabetes mellitus: a patient is
diagnosed in the early stages of non-insulin dependent diabetes
mellitus. A peptide as described herein is formulated in an enteric
capsule. The patient is directed to take one tablet after each meal
for the purpose of stimulating the insulin signaling pathway, and
thereby controlling glucose metabolism to levels that obviate the
need for administration of exogenous insulin
[0268] In another example, the method according to these
embodiments of the present invention can be used to treat affective
disorders such as unipolar disorders (e.g., depression) and bipolar
disorders (e.g., manic depression). As is demonstrated herein, the
effect of the peptides as described herein was exemplified on
up-regulation of .beta.-catenin levels, thus indicating, a role of
these GSK-3 inhibitors in the treatment of affective disorders.
[0269] As GSK-3 is also considered to be an important player in the
pathogenesis of neurodegenerative disorders and diseases, the
method according to this aspect of the present invention can be
further used to treat a variety of such disorders and diseases.
[0270] In one example, since inhibition of GSK-3 results in halting
neuronal cell death, the method according to these embodiments of
the present invention can be used to treat a neurodegenerative
disorder that results from an event that cause neuronal cell death.
Such an event can be, for example, cerebral ischemia, stroke,
traumatic brain injury or bacterial infection.
[0271] In another example, since GSK-3 activity is implicated in
various central nervous system disorders and neurodegenerative
diseases, the method according to these embodiments can be used to
treat various chronic neurodegenerative diseases such as, but not
limited to, Alzheimer's disease, Huntington's disease, Parkinson's
disease, AIDS associated dementia, amyotrophic lateral sclerosis
(AML) and multiple sclerosis.
[0272] As is discussed hereinabove, GSK-3 activity has particularly
been implicated in the pathogenesis of Alzheimer's disease. Hence,
in one representative embodiment of the method described herein,
there is provided a method of treating a patient with Alzheimer's
disease: A patient diagnosed with Alzheimer's disease is
administered with a peptide as described herein, which inhibits
GSK-3-mediated tau hyperphosphorylation, prepared in a formulation
that crosses the blood brain barrier (BBB). The patient is
monitored for tau phosphorylated polymers by periodic analysis of
proteins isolated from the patient's brain cells for the presence
of phosphorylated forms of tau on an SDS-PAGE gel known to
characterize the presence of and progression of the disease. The
dosage of the peptide is adjusted as necessary to reduce the
presence of the phosphorylated forms of tau protein.
[0273] GSK-3 has also been implicated with respect to psychotic
disorders such as schizophrenia, and therefore the method according
to this aspect of embodiments of the present invention can be
further used to treat psychotic diseases or disorders, such as
schizophrenia.
[0274] GSK-3 has also been implicated with respect to affective
disorders. Therefore, in another example, the method according to
this aspect of the present invention can be used to treat affective
disorders such as unipolar disorders (e.g., depression) and bipolar
disorders (e.g., manic depression).
[0275] It should be noted that the peptides described herein are
particularly advantageous in the treatment of psychotic, affective
and neurodegenerative diseases or disorders since, apart from
exerting enhanced inhibition activity of GSK-3, it is postulated
that the inclusion of multiple hydrophobic amino acid residues
within the peptides further provides for enhanced lipophilicity of
the peptides and, as a result, for enhanced permeability through
the blood brain barrier (BBB). This enhanced permeability may allow
a systemic, rather than local, administration of the peptides, such
that the need to administer the inhibitors intracerebroventicularly
(icy) is avoided.
[0276] GSK-3 has also been implicated with respect to
cardiovascular conditions, and therefore, the peptides described
herein can be further used to treat cardiovascular diseases or
disorders.
[0277] Cardiovascular diseases and disorders include, but are not
limited to, atherosclerosis, a cardiac valvular disease, stenosis,
restenosis, in-stent-stenosis, myocardial infarction, coronary
arterial disease, acute coronary syndromes, congestive heart
failure, angina pectoris, myocardial ischemia, thrombosis,
Wegener's granulomatosis, Takayasu's arteritis, Kawasaki syndrome,
anti-factor VIII autoimmune disease or disorder, necrotizing small
vessel vasculitis, microscopic polyangiitis, Churg and Strauss
syndrome, pauci-immune focal necrotizing glomerulonephritis,
crescentic glomerulonephritis, antiphospholipid syndrome, antibody
induced heart failure, thrombocytopenic purpura, autoimmune
hemolytic anemia, cardiac autoimmunity, Chagas' disease or
disorder, and anti-helper T lymphocyte autoimmunity.
[0278] GSK-3 has also been implicated with respect to conditions
(e.g., infections) associated with pathogenic parasites (e.g.,
malaria and trypanosomiasis), and therefore, the peptides described
herein can be further used to treat a condition (e.g., infection)
that is associated with a presence of a pathogenic parasite in a
subject. Exemplary parasites include Acanthamoeba, Anisakis,
Ascaris lumbricoides, Botfly, Balantidium coli, Bedbug, Cestoda
(tapeworm), Chiggers, Cochliomyia hominivorax, Entamoeba
histolytica, Fasciola hepatica, Giardia lamblia, Hookworm,
Leishmania, Linguatula serrata, Liver fluke, Loa loa,
Paragonimus--lung fluke, Pinworm, Schistosoma, Strongyloides
stercoralis, Mites, Tapeworm, Toxoplasma gondii, Trypanosoma,
Whipworm, Wuchereria bancrofti and Plasmodium falciparum and
related malaria-causing protozoan parasites.
[0279] Exemplary conditions caused by pathogenic parasites include,
but are not limited to, Acanthamoeba keratitis, Amoebiasis,
Ascariasis, Babesiosis, Balantidiasis, Baylisascariasis, Chagas
disease, Clonorchiasis, Cochliomyia, Cryptosporidiosis,
Diphyllobothriasis, Dracunculiasis (caused by the Guinea worm),
Echinococcosis, Elephantiasis, Enterobiasis, Fascioliasis,
Fasciolopsiasis, Filariasis, Giardiasis, Gnathostomiasis,
Hymenolepiasis, Isosporiasis, Katayama fever, Leishmaniasis, Lyme
disease, Malaria, Metagonimiasis, Myiasis, Onchocerciasis,
Pediculosis, Scabies, Schistosomiasis, Sleeping sickness,
Strongyloidiasis, Taeniasis (cause of Cysticercosis), Toxocariasis,
Toxoplasmosis, Trichinosis and Trichuriasis.
[0280] GSK-3 has also been suggested to be involved in stem cell
maintenance and/or differentiation. Accordingly, the peptides
described herein can be further utilized in the treatment of
conditions in which transplantation of stem cells is used as part
of the treatment. Such conditions include, for example, cancer and
damaged tissues (treatable by tissue regeneration).
[0281] In some embodiments, the peptides described herein can be
utilized for maintaining and/or differentiating stem cells. Thus,
in some embodiments, there is provided a method of maintaining
and/or differentiating stem cells, which is effected by contacting
a peptide as described herein with stem cells. In some embodiments,
the contacting is effected ex-vivo. In some embodiments, the
contacting is effected in the presence of a physiological medium,
as acceptable for stem cells preparations. In some embodiments, the
contacting is effected by placing stem cells in a suitable medium
which further comprises a peptide as described herein.
[0282] The method according to this aspect of the present invention
can be further effected by co-administering to the subject one or
more additional active ingredient(s) which is capable of altering
an activity of GSK-3.
[0283] As used herein, "co-administering" describes administration
of a peptide as described herein in combination with the additional
active ingredient(s) (also referred to herein as active or
therapeutic agent). The additional active agent can be any
therapeutic agent useful for treatment of the patient's condition.
The co-administration may be simultaneous, for example, by
administering a mixture of the peptide and the additional
therapeutic agent, or may be accomplished by administration of the
peptide and the active agent separately, such as within a short
time period. Co-administration also includes successive
administration of the peptide and one or more of another
therapeutic agent. The additional therapeutic agent or agents may
be administered before or after the peptide. Dosage treatment may
be a single dose schedule or a multiple dose schedule.
[0284] An example of an additional active agent is insulin.
[0285] Preferably, the additional active agent is capable of
inhibiting an activity of GSK-3, such that the additional active
agent can be any GSK-3 inhibitor other than the peptides described
herein, and thus can be, as non-limiting examples, lithium,
valproic acid and other peptides or small molecules that are shown
to inhibit GSK-3 activity as described herein.
[0286] Alternatively, the additional active agent can be an agent
that is capable of downregulating an expression of GSK-3.
[0287] An agent that downregulates GSK-3 expression refers to any
agent which affects GSK-3 synthesis (decelerates) or degradation
(accelerates) either at the level of the mRNA or at the level of
the protein. For example, a small interfering polynucleotide
molecule which is designed to downregulate the expression of GSK-3
can be used as an additional active agent according to some
embodiments of the present invention.
[0288] An example for a small interfering polynucleotide molecule
which can downregulate the expression of GSK-3 is a small
interfering RNA or siRNA, such as, for example, the morpholino
antisense oligonucleotides described by in Munshi et al. (Munshi C
B, Graeff R, Lee H C, J Biol Chem 2002 Dec. 20; 277(51):49453-8),
which includes duplex oligonucleotides which direct sequence
specific degradation of mRNA through the previously described
mechanism of RNA interference (RNAi) (Hutvagner and Zamore (2002)
Curr. Opin. Genetics and Development 12:225-232).
[0289] As used herein, the phrase "duplex oligonucleotide" refers
to an oligonucleotide structure or mimetics thereof, which is
formed by either a single self-complementary nucleic acid strand or
by at least two complementary nucleic acid strands. The "duplex
oligonucleotide" of the present invention can be composed of
double-stranded RNA (dsRNA), a DNA-RNA hybrid, single-stranded RNA
(ssRNA), isolated RNA (i.e., partially purified RNA, essentially
pure RNA), synthetic RNA and recombinantly produced RNA.
[0290] Preferably, the specific small interfering duplex
oligonucleotide of the present invention is an oligoribonucleotide
composed mainly of ribonucleic acids.
[0291] Instructions for generation of duplex oligonucleotides
capable of mediating RNA interference are provided in
wwwdotambiondotcom.
[0292] Hence, the small interfering polynucleotide molecule
according to some embodiments of the present invention can be an
RNAi molecule (RNA interference molecule).
[0293] Alternatively, a small interfering polynucleotide molecule
can be an oligonucleotide such as a GSK-3-specific antisense
molecule or a rybozyme molecule, further described hereinunder.
[0294] Antisense molecules are oligonucleotides, which contain two
or more chemically distinct regions, each made up of at least one
nucleotide. These oligonucleotides typically contain at least one
region wherein the oligonucleotide is modified so as to confer upon
the oligonucleotide increased resistance to nuclease degradation,
increased cellular uptake, and/or increased binding affinity for
the target polynucleotide. An additional region of the
oligonucleotide may serve as a substrate for enzymes capable of
cleaving RNA:DNA or RNA:RNA hybrids. An example for such includes
RNase H, which is a cellular endonuclease which cleaves the RNA
strand of an RNA:DNA duplex. Activation of RNase H, therefore,
results in cleavage of the RNA target, thereby greatly enhancing
the efficiency of oligonucleotide inhibition of gene expression.
Consequently, comparable results can often be obtained with shorter
oligonucleotides when chimeric oligonucleotides are used, compared
to phosphorothioate deoxyoligonucleotides hybridizing to the same
target region. Cleavage of the RNA target can be routinely detected
by gel electrophoresis and, if necessary, associated nucleic acid
hybridization techniques known in the art.
[0295] The antisense molecules of the present invention may be
formed as composite structures of two or more oligonucleotides,
modified oligonucleotides, as described above. Representative U.S.
patents that teach the preparation of such hybrid structures
include, but are not limited to, U.S. Pat. Nos. 5,013,830;
5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133;
5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of
which is herein fully incorporated by reference.
[0296] Rybozyme molecules are being increasingly used for the
sequence-specific inhibition of gene expression by the cleavage of
mRNAs. Several rybozyme sequences can be fused to the
oligonucleotides of the present invention. These sequences include
but are not limited ANGIOZYME specifically inhibiting formation of
the VEGF-R (Vascular Endothelial Growth Factor receptor), a key
component in the angiogenesis pathway, and HEPTAZYME, a rybozyme
designed to selectively destroy Hepatitis C Virus (HCV) RNA,
(Rybozyme Pharmaceuticals, Incorporated--WEB home page).
[0297] Further alternatively, a small interfering polynucleotide
molecule, according to the present invention can be a DNAzyme.
[0298] DNAzymes are single-stranded catalytic nucleic acid
molecules. A general model (the "10-23" model) for the DNAzyme has
been proposed. "10-23" DNAzymes have a catalytic domain of 15
deoxyribonucleotides, flanked by two substrate-recognition domains
of seven to nine deoxyribonucleotides each. This type of DNAzyme
can effectively cleave its substrate RNA at purine:pyrimidine
junctions (Santoro, S. W. & Joyce, G. F. Proc. Natl, Acad. Sci.
USA 199; for rev of DNAzymes see Khachigian, L M Curr Opin Mol Ther
2002; 4:119-21).
[0299] Examples of construction and amplification of synthetic,
engineered DNAzymes recognizing single and double-stranded target
cleavage sites have been disclosed in U.S. Pat. No. 6,326,174 to
Joyce et al. DNAzymes of similar design directed against the human
Urokinase receptor were recently observed to inhibit Urokinase
receptor expression, and successfully inhibit colon cancer cell
metastasis in vivo (Itoh et al., 20002, Abstract 409, Ann Meeting
Am Soc Gen Ther wwwdotasgtdotorg). In another application, DNAzymes
complementary to bcr-ab1 oncogenes were successful in inhibiting
the oncogenes expression in leukemia cells, and lessening relapse
rates in autologous bone marrow transplant in cases of CML and
ALL.
[0300] Oligonucleotides designed according to the teachings of the
present invention can be generated according to any oligonucleotide
synthesis method known in the art such as enzymatic synthesis or
solid phase synthesis. Equipment and reagents for executing
solid-phase synthesis are commercially available from, for example,
Applied Biosystems. Any other means for such synthesis may also be
employed; the actual synthesis of the oligonucleotides is well
within the capabilities of one skilled in the art.
[0301] Further according to embodiments of the present invention
there is provided a use of the peptides as described herein in the
manufacture of a medicament for treating a biological condition
associated with GSK-3 activity, as described herein.
[0302] Further according to embodiments of the present invention
there is provided a peptide as described herein, which is
identified for use in the treatment of a biological condition
associated with GSK-3 activity, as described herein.
[0303] In any of the methods and uses described herein, the
peptides described herein can be utilized in combination with one
or more additional active ingredient(s) or agent(s) which is
capable of altering an activity of GSK-3, as described herein.
[0304] In any of the methods and uses described herein the peptide
described herein can be utilized either per se, or, preferably, the
peptide forms a part of a pharmaceutical composition, which may
further comprise a pharmaceutically acceptable carrier.
[0305] As used herein a "pharmaceutical composition" refers to a
preparation of one or more of the peptides described herein (as
active ingredient), or physiologically acceptable salts or prodrugs
thereof, with other chemical components including but not limited
to physiologically suitable carriers, excipients, lubricants,
buffering agents, antibacterial agents, bulking agents (e.g.
mannitol), antioxidants (e.g., ascorbic acid or sodium bisulfite),
anti-inflammatory agents, anti-viral agents, chemotherapeutic
agents, anti-histamines and the like. The purpose of a
pharmaceutical composition is to facilitate administration of a
compound to a subject.
[0306] The term "active ingredient", which is also referred to
herein interchangeably as "active agent" refers to a compound,
which is accountable for a biological effect.
[0307] The terms "physiologically acceptable carrier" and
"pharmaceutically acceptable carrier" which may be interchangeably
used refer to a carrier or a diluent that does not cause
significant irritation to an organism and does not abrogate the
biological activity and properties of the administered
compound.
[0308] Herein the term "excipient" refers to an inert substance
added to a pharmaceutical composition to further facilitate
administration of a drug. Examples, without limitation, of
excipients include calcium carbonate, calcium phosphate, various
sugars and types of starch, cellulose derivatives, gelatin,
vegetable oils and polyethylene glycols.
[0309] Techniques for formulation and administration of drugs may
be found in "Remington's Pharmaceutical Sciences" Mack Publishing
Co., Easton, Pa., latest edition, which is incorporated herein by
reference.
[0310] Pharmaceutical compositions for use in accordance with the
present invention thus may be formulated in conventional manner
using one or more pharmaceutically acceptable carriers comprising
excipients and auxiliaries, which facilitate processing of the
compounds into preparations which can be used pharmaceutically.
Proper formulation is dependent upon the route of administration
chosen. The dosage may vary depending upon the dosage form employed
and the route of administration utilized. The exact formulation,
route of administration and dosage can be chosen by the individual
physician in view of the patient's condition (see e.g., Fingl et
al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.
1).
[0311] The pharmaceutical composition may be formulated for
administration in either one or more of routes depending on whether
local or systemic treatment or administration is of choice, and on
the area to be treated. Administration may be done orally, nasally,
by inhalation, or parenterally, for example by intravenous drip or
intraperitoneal, subcutaneous, intramuscular or intravenous
injection, or topically (including ophtalmically, vaginally,
rectally and intranasally).
[0312] Formulations for topical administration may include but are
not limited to lotions, ointments, gels, creams, suppositories,
drops, liquids, sprays and powders. Conventional pharmaceutical
carriers, aqueous, powder or oily bases, thickeners and the like
may be necessary or desirable.
[0313] Compositions for oral administration include powders or
granules, suspensions or solutions in water or non-aqueous media,
sachets, pills, caplets, capsules or tablets. Thickeners, diluents,
flavorings, dispersing aids, emulsifiers or binders may be
desirable.
[0314] Formulations for parenteral administration may include, but
are not limited to, sterile solutions which may also contain
buffers, diluents and other suitable additives. Slow release
compositions are envisaged for treatment.
[0315] In some embodiments, there is provided a pharmaceutical
composition, as described herein, being formulated for nasal
administration, as defined herein.
[0316] The amount of a composition to be administered will, of
course, be dependent on the subject being treated, the severity of
the affliction, the manner of administration, the judgment of the
prescribing physician, etc.
[0317] The pharmaceutical composition may further comprise
additional pharmaceutically active or inactive agents such as, but
not limited to, an anti-bacterial agent, an antioxidant, a
buffering agent, a bulking agent, a surfactant, an
anti-inflammatory agent, an anti-viral agent, a chemotherapeutic
agent and an anti-histamine.
[0318] According to an embodiment of the present invention, the
pharmaceutical composition described herein is packaged in a
packaging material and identified in print, in or on the packaging
material, for use in the treatment of a medical condition
associated with GSK-3 activity, as described herein.
[0319] Compositions of the present invention may, if desired, be
presented in a pack or dispenser device, such as an FDA approved
kit, which may contain one or more unit dosage forms containing the
active ingredient. The pack may, for example, comprise metal or
plastic foil, such as a blister pack. The pack or dispenser device
may be accompanied by instructions for administration. The pack or
dispenser may also be accommodated by a notice associated with the
container in a form prescribed by a governmental agency regulating
the manufacture, use or sale of pharmaceuticals, which notice is
reflective of approval by the agency of the form of the
compositions or human or veterinary administration. Such notice,
for example, may be of labeling approved by the U.S. Food and Drug
Administration for prescription drugs or of an approved product
insert.
[0320] In some embodiments, the pharmaceutical composition is
identified for use in combination with an additional active agent,
as described herein.
[0321] In some embodiments, the pharmaceutical composition further
comprises an additional active agent as described herein, being
co-formulated with the peptide as described herein.
[0322] Further according to embodiments of the present invention
there is provided a use of any of the peptides and/or the GSK-3
substrate competitive inhibitors as described herein in the
manufacture of a medicament for treating a biological condition
associated with GSK-3 activity, as described herein.
[0323] Further according to embodiments of the present invention
there is provided a method of treating a biological condition
associated with GSK-3 activity, as described herein, an/or of
inhibiting a GSK-3 activity, which is effected by administering to
a subject in need thereof any of the GSK-3 substrate competitive
inhibitors as described herein.
[0324] Further according to embodiments of the present invention
there is provided a GSK-3 substrate competitive inhibitor and/or a
peptide as described herein, which is identified for use in the
treatment of a biological condition associated with GSK-3 activity,
as described herein.
[0325] In any of the methods and uses described herein the GSK-3
substrate competitive inhibitors as described herein can be
utilized either per se, or, preferably, or forms a part of a
pharmaceutical composition, which may further comprise a
pharmaceutically acceptable carrier, as described herein.
[0326] As described herein, in the course of the studies conducted
for designing GSK-3 substrate competitive inhibitors with improved
performance, the present inventors have prepared various mutants of
GSK-3.beta.. Such mutants served as a tool for identifying
potential inhibitors of GSK-3 activity.
[0327] These mutants included pre-determined modifications at a
subunit of the catalytic binding site of GSK-3, previously
described as the 89-95 loop, as set forth in SEQ ID NO:2.
[0328] Thus, according to an aspect of some embodiments of the
present invention there is provided a polypeptide which comprises
an amino acid sequence of a mutated GSK-3 enzyme, wherein an amino
acid sequence of mutated GSK-3 enzyme comprises at least one amino
acid substitution with respect to an amino acid sequence of a
catalytic binding site of a wild-type GSK-3.
[0329] The "mutated GSK-3 enzyme" of these embodiments of the
present invention refers to a polypeptide which differs from a
corresponding wild-type GSK-3 (i.e., the starting point GSK-3) by
at least one mutation (e.g., substitution).
[0330] A wild-type GSK-3 is as defined hereinabove for GSK-3.
[0331] In some embodiments, the mutated GSK-3 enzyme is
characterized by a substrate specificity which is substantially
identical to that of a respective wild-type GSK-3.
[0332] According to some embodiments, the mutated enzyme comprises
at least one amino acid substitution with respect to an amino acid
sequence of a subunit of the catalytic (substrate's) binding site
of the corresponding wild-type GSK-3.
[0333] According to some embodiments of the present invention, the
subunit of the substrate's binding site of a wild-type GSK-3
comprises positions 89-95 of the amino acid sequence of the
wild-type GSK-3, and has an amino acid sequence as set forth in SEQ
ID NO:2. This subunit is also referred to herein as a 89-95 subunit
or a 89-95 loop.
[0334] According to some embodiments of the present invention, the
amino acid substitution is at one or more of positions 89, 90, 91,
92, 93, 94 and/or 95 of the 89-95 subunit. In most of the living
organisms expressing GSK-3, these positions correspond to Q89
(Gln89), R92 (Arg92), F93 (Phe93), K94 (Lys94), and N95 (Asn95), as
is in e.g., human GSK-3.beta.. Thus, according to an aspect of some
embodiments of the present invention there is provided a
polypeptide which comprises an amino acid sequence of a mutated
GSK-3 enzyme, wherein an amino acid sequence of the mutated GSK-3
enzyme comprises at least one amino acid substitution with respect
to position Gln89, Asp90, Lys91, Arg92, Phe93, Lys94 and/or Asn95
of a corresponding wild-type GSK-3 (e.g., having an amino acid
sequence as set forth in SEQ ID NO:1).
[0335] Thus, according to an aspect of some embodiments of the
present invention there is provided a polypeptide which comprises
an amino acid sequence of a mutated GSK-3 enzyme, wherein an amino
acid sequence of the mutated GSK-3 enzyme comprises an amino acid
substitution with respect to position Val214, or an equivalent
thereof of a corresponding wild-type GSK-3 (e.g., having an amino
acid sequence as set forth in SEQ ID NO:1).
[0336] Herein throughout, whenever a three-letter abbreviation of
an amino acid is followed by a number it is meant the number of the
indicated amino acid residue along the amino acid sequence
downstream the N-terminus of the enzyme. The three-letter
abbreviations described herein are as commonly used in the art.
[0337] By "position" it is meant a coordinate of the amino acid,
whereby the indicated coordinate encompasses also an amino acid
equivalent, as defined herein.
[0338] According to some embodiments of the present invention, the
amino acid substitution comprises an alanine substitution such that
one or more of the amino acids at positions 89, 92, 93, 94 and 95,
or at position 214 of a wild-type GSK-3 is substituted by an
alanine residue. However, substitution by any other amino acid
residue is also contemplated.
[0339] The mutated GSK-3 enzyme is thus characterized by at least
one amino acid substitution with respect to an amino acid sequence
of a corresponding wild type GSK-3.
[0340] Exemplary polypeptides according to some embodiments of the
present invention comprise an amino acid sequence at positions
89-95 as set forth in SEQ ID NOS:6-10 and 47.
[0341] The present embodiments also encompass functional homologues
of the polypeptides described herein, such homologues can be at
least 50%, at least 55%, at least 60%, at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 95% or more say
100% homologous to SEQ ID NOS:6-10 and 47, as long as the indicated
substitution is maintained.
[0342] In some embodiments, the polypeptides described herein are
phosphorylated. In some embodiments, the polypeptides described
herein comprise an amino acid sequence in which Tyr 216 or an
equivalent thereof is phosphorylated.
[0343] Recombinant techniques, as described herein, are preferably
used to generate the polypeptides of the present invention.
Alternatively, the polypeptides are prepared by chemical synthesis,
using, for example, solid phase synthesis as described herein.
[0344] Thus, further according to an aspect of some embodiments of
the present invention there is provided a polynucleotide encoding
the isolated polypeptide as described herein.
[0345] As used herein the term "polynucleotide" refers to a single
or a double stranded nucleic acid sequence which is isolated and
provided in the form of an RNA sequence, a complementary
polynucleotide sequence (cDNA), a genomic polynucleotide sequence
and/or a composite polynucleotide sequences (e.g., a combination of
the above).
[0346] As used herein the phrase "complementary polynucleotide
sequence" refers to a sequence, which results from reverse
transcription of messenger RNA using a reverse transcriptase or any
other RNA dependent DNA polymerase. Such a sequence can be
subsequently amplified in vivo or in vitro using a DNA dependent
DNA polymerase.
[0347] As used herein the phrase "genomic polynucleotide sequence"
refers to a sequence derived (isolated) from a chromosome and thus
it represents a contiguous portion of a chromosome.
[0348] As used herein the phrase "composite polynucleotide
sequence" refers to a sequence, which is at least partially
complementary and at least partially genomic. A composite sequence
can include some exonal sequences required to encode the
polypeptide of the present invention, as well as some intronic
sequences interposing therebetween. The intronic sequences can be
of any source, including of other genes, and typically will include
conserved splicing signal sequences. Such intronic sequences may
further include cis acting expression regulatory elements.
[0349] According to some embodiments, the nucleic acid sequence
which encodes the mutated GSK-3 enzyme is of a mammalian origin,
such as a mouse origin, a human origin, a rat origin, a rabbit
origin or a combination thereof (e.g., a result of gene shuffling),
and is preferably of a human origin.
[0350] To produce a polypeptide of the present invention using
recombinant technology, a polynucleotide encoding the polypeptide
as described herein is ligated into a nucleic acid expression
construct, which includes the polynucleotide sequence under the
transcriptional control of a promoter sequence suitable for
directing constitutive or inducible transcription in the host
cells, as further described hereinbelow.
[0351] Other than containing the necessary elements for the
transcription and translation of the inserted coding sequence, the
expression construct of the present invention can also include
sequences (i.e., tags) engineered to enhance stability, production,
purification, yield or toxicity of the expressed polypeptide. Such
a fusion protein can be designed so that the fusion protein can be
readily isolated by affinity chromatography; e.g., by
immobilization on a column specific for the heterologous protein.
Where a cleavage site is engineered between the peptide moiety and
the heterologous protein, the peptide can be released from the
chromatographic column by treatment with an appropriate enzyme or
agent that disrupts the cleavage site [e.g., see Booth et al.
(1988) Immunol. Lett. 19:65-70; and Gardella et al., (1990) J.
Biol. Chem. 265:15854-15859].
[0352] A variety of prokaryotic or eukaryotic cells can be used as
host-expression systems to express the peptide coding sequence.
These include, but are not limited to, microorganisms, such as
bacteria transformed with a recombinant bacteriophage DNA, plasmid
DNA or cosmid DNA expression vector containing the peptide coding
sequence; yeast transformed with recombinant yeast expression
vectors containing the polypeptide coding sequence; plant cell
systems infected with recombinant virus expression vectors (e.g.,
cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or
transformed with recombinant plasmid expression vectors, such as Ti
plasmid, containing the polypeptide coding sequence. Mammalian
expression systems can also be used to express the peptides of the
present invention. Bacterial systems are preferably used to produce
recombinant polypeptides, according to the present invention,
thereby enabling a high production volume at low cost.
[0353] Other expression systems such as insects and mammalian host
cell systems, which are well known in the art can also be used by
the present invention.
[0354] In any case, transformed cells are cultured under effective
conditions, which allow for the expression of high amounts of
recombinant polypeptides. Effective culture conditions include, but
are not limited to, effective media, bioreactor, temperature, pH
and oxygen conditions that permit protein production. An effective
medium refers to any medium in which a cell is cultured to produce
the recombinant peptides of the present invention. Such a medium
typically includes an aqueous solution having assimilable carbon,
nitrogen and phosphate sources, and appropriate salts, minerals,
metals and other nutrients, such as vitamins. Cells of the present
invention can be cultured in conventional fermentation bioreactors,
shake flasks, test tubes, microtiter dishes, and petri plates.
Culturing can be carried out at a temperature, pH and oxygen
content appropriate for a recombinant cell. Such culturing
conditions are within the expertise of one of ordinary skill in the
art (see Example 1 of the Examples section).
[0355] Depending on the vector and host system used for production,
resultant proteins of the present invention may either remain
within the recombinant cell; be secreted into the fermentation
medium; be secreted into a space between two cellular membranes,
such as the periplasmic space in E. coli; or be retained on the
outer surface of a cell or viral membrane.
[0356] Following a certain time in culture, recovery of the
recombinant protein is effected. The phrase "recovering the
recombinant protein" refers to collecting the whole fermentation
medium containing the protein and need not imply additional steps
of separation or purification. Proteins of the present invention
can be purified using a variety of standard protein purification
techniques, such as, but not limited to, affinity chromatography,
ion exchange chromatography, filtration, electrophoresis,
hydrophobic interaction chromatography, gel filtration
chromatography, reverse phase chromatography, concanavalin A
chromatography, chromatofocusing and differential
solubilization.
[0357] Thus, further according to embodiments of the present
invention there is provided a nucleic acid construct comprising the
polynucleotide as described herein.
[0358] Further according to embodiments of the present invention
there is provided a host cell system comprising the nucleic acid
construct as described herein.
[0359] The polypeptides described herein can be utilized in a
screening method for identifying putative GSK-3 substrate
competitive inhibitors. Since it has been demonstrated herein that
the mutated polypeptides lack those coordinates that provide for
increased binding to the substrate's binding site in GSK-3, it is
suggested that when a candidate inhibitor exhibits a reduced effect
on the activity of a mutated enzyme, compared to its effect on a
corresponding wild-type GSK-3, such a candidate is characterized by
strong affinity to the lacking coordinate and thus could serve as a
potent substrate competitive inhibitor. It is to be noted that
potent substrate competitive inhibitors should not only exhibit
binding to the substrate's binding site of the enzyme, but should
exhibit such a binding that is at least similar, and preferably
stronger than that of a GSK-3 substrate.
[0360] Thus, according to an aspect of some embodiments of the
present invention there is provided a method of identifying a
putative GS K-3 substrate competitive inhibitor. In some
embodiments, the method is effected by screening a plurality of
substances for a substance which exhibits inhibition of at least
20% of an activity of a wild-type GSK-3 enzyme and which exhibits
inhibition of no more than 20% of an activity of the polypeptide
which comprises an amino acid sequence of a mutated GSK-3, as
described herein.
[0361] By "putative" it is meant capable of reducing an activity of
a GSK-3 enzyme by at least 10%, at least 20%, at least 30%, at
least 40%, at least 50%, and so forth, up to 100%, including any
intermediate value, as determined by methods known in the art for
determining a catalytic activity of a kinase, and as if further
detailed hereinbelow. In some embodiments, reducing the activity of
a GSK-3 by the substance is effected by competing with the
substrate on the binding to the catalytic site of the enzyme.
Determining a substrate competitive nature of an inhibitor can be
performed by methods known in the art.
[0362] In some embodiments, the screening is effected by
determining a level of inhibition of a wild-type GSK-3 enzyme by
each of the tested substance; determining a level of inhibition of
a polypeptide which comprises a mutated GSK-3 enzyme as described
herein by each of the tested substance; and comparing these levels
of inhibition for each substance. Those substances that
substantially inhibit an activity of a wild-type GSK-3 but
substantially lack an inhibition activity of a mutated GSK-3, as in
the polypeptides described herein, are considered putative GSK-3
substrate competitive inhibitors.
[0363] By "substantially inhibiting an activity of a wild-type
GSK-3" it is meant that a phosphorylation of a GSK-3 substrate in
the presence of the tested substance is lower than the
phosphorylation in the absence of the tested compound by at least
20%, or at least 30%, or at least 40%, or at least 50%, or at least
60%, or at least 70%, or at least 80%, or at least 90 5, and even
by 100%.
[0364] By substantially lacking an inhibition activity it is meant
that a phosphorylation of a mutated GSK-3 substrate in the presence
of the tested substance is substantially the same as the
phosphorylation in the absence of the tested compound, or is lower
by no more than 1%, or 5%, or 10%, or 12%, or 15% and in any case
by no more than 20%.
[0365] In some embodiments, a substance identified as a putative
inhibitor exhibits inhibition of at least 50% of a wild-type GSK-3
enzyme and an inhibition of less than 10% of an activity of a
mutated GSK-3 enzyme.
[0366] Determining an activity of a GSK-3 enzyme or of a mutant
thereof can be performed by any method known in the art for
assaying a kinase activity (e.g., phosphorylation). In some
embodiments an activity of a GSK-3 enzyme is determined by
contacting the enzyme with a GSK-3 substrate, as described herein,
in a suitable medium, and determining a level of a phosphorylated
substrate thereafter. An inhibition activity of a substance can be
readily determined by contacting the enzyme with a GSK-3 substrate
and with the tested substance, as described herein, in a suitable
medium, and determining a level of a phosphorylated substrate
thereafter. The ratio (e.g., percentage) between the level of
phosphorylation of the substrate in the presence and absence of the
tested compound is indicative of the level of inhibition exhibited
by the tested substance.
[0367] Similarly, according to another aspect of some embodiments
of the present invention there is provided a method of identifying
a putative substrate competitive inhibitor of GSK-3, which is
effected by screening a plurality of substances for a substance
which exhibits inhibition of at least 20% of an activity of a
wild-type GSK-3 enzyme and which exhibits inhibition of less than
20% of said activity of a mutated GSK-3 enzyme, wherein the mutated
GSK-3 enzyme comprises at least one amino acid substitution with
respect to position Asp90, Lys91, Arg92, Phe93 and/or Lys94 or to
position Val214 of a corresponding wild-type GSK3 enzyme, as
described herein, and, for example, as set forth in SEQ ID NOS:6-10
and 47.
[0368] In some embodiments, the mutated GSK-3 enzyme comprises at
least one amino acid substitution with respect to position Asp90,
Arg92, Phe93 and/or Lys94 of a corresponding wild-type GSK3 enzyme,
for example, as set forth in SEQ ID NOS:6 AND 8-10).
[0369] In some embodiments, the mutated GSK-3 enzyme comprises an
amino acid substitution with respect to position Phe93 of said
corresponding wild-type GSK-3 enzyme, for example, as set forth in
SEQ ID NO:9.
[0370] In any of the screening methods described herein, the tested
substances can be peptides, polypeptides and/or small organic
molecules, as defined herein.
[0371] The experimental identification of Phe93 as an important
binding site within the substrate's binding subunit of a GSK-3
enzyme, can be further utilized in in silico screening for a
putative substrate competitive inhibitor.
[0372] Thus, according to an aspect of some embodiments of the
present invention there is provided another method of identifying a
putative substrate competitive inhibitor of GSK-3. In some
embodiments, this method is effected by screening a plurality of
substances for a substance capable of interacting with a Phe93
residue, or an equivalent amino acid thereof, in a catalytic
binding site of a GSK-3 enzyme.
[0373] In some embodiments, the method is effected by determining
the binding of a candidate substance to Phe93 or an equivalent
thereof in GSK-3 by comparing the inhibition of an activity of a
wild-type GSK-3 as exhibited by the substance to an inhibition of
an activity of a mutated GSK-3 that comprises an amino acid
substitution with respect to Phe93, as described herein, as
exhibited by the substance.
[0374] Such a method is effected as described hereinabove, while
utilizing, for example, a polypeptide that comprises a mutated
enzyme as set forth in SEQ ID NO:9.
[0375] In some embodiments, the method comprises computationally
screening the plurality of substances for a substance capable of
interacting with phenylalanine at coordinate 93, or with an
equivalent amino acid thereof, within a set of atomic structural
coordinates defining a three-dimensional atomic structure of a
catalytic binding site of GSK-3 (e.g., a GSK-3 as set forth in SEQ
ID NO:1).
[0376] In some embodiments, the method is further effected by
identifying a substance that is further capable, in addition to
interacting with Phe93, of interacting with at least one additional
amino acid within the catalytic binding site of a GSK-3.
[0377] In some embodiments, the additional amino acid is one or
more of Phe67, Gln89, Asp90, Lys91, Arg92, Lys94 and Asp95 in a
GSK-3 enzyme (e.g., wild-type GSK-3 enzyme such as human
GSK-3.beta.).
[0378] In some embodiments, the additional amino acid is one or
more of Phe67, Gln89, Asp90, Arg92, Lys 94 and Asp95 in a GSK-3
enzyme (e.g., wild-type GSK-3 enzyme such as human
GSK-3.beta.).
[0379] In some embodiments, the additional amino acid is one or
more of Phe67, Gln89 and Asn95 (e.g., wild-type GSK-3 enzyme such
as human GSK-3.beta.).
[0380] In some embodiments, the additional amino acid is one or
more of any of the amino acids identified hitherto with respect to
substrate's binding in GSK-3, as delineated hereinabove.
[0381] The method of these embodiments of the present invention is
generally effected by constructing a model using a set of atomic
structural coordinates defining a three-dimensional atomic
structure of GSK-3 and computationally screening a plurality of
substances, as described herein, for a substance capable of
interacting with Phe93, to thereby identify the GSK-3
inhibitor.
[0382] Typically, obtaining the set of atomic coordinates which
define the three dimensional structure of an enzyme can be effected
using various approaches which are well known in the art.
[0383] Structural data obtained is preferably recorded on a
computer readable medium so as to enable data manipulation and
construction of computational models. As used herein, "computer
readable medium" refers to any medium which can be read and
accessed directly by a computer. Such media include, but are not
limited to, magnetic storage media, such as floppy discs, hard disc
storage medium, and magnetic tape; optical storage media such as
optical discs or CD-ROM; electrical storage media such as RAM and
ROM; and hybrids of these categories such as magnetic/optical
storage media. Selection and use of appropriate storage media is
well within the capabilities of one of ordinary skill in the
art.
[0384] As used herein, "recorded" refers to a process of storing
information on computer readable medium.
[0385] It will be appreciated that a number of data storage devices
can be used for creating a computer readable medium having recorded
thereon the structural data of the present invention. The choice of
the data storage structure is typically based on the means chosen
to access the stored information. In addition, a variety of data
processor programs and formats can be used to store the data
information of the present invention on computer readable medium.
The sequence information can be represented in a word processing
text file, formatted in commercially-available software such as
WordPerfect and MICROSOFT Word, or represented in the form of an
ASCII file, stored in a database application, such as DB2, Sybase,
Oracle, or the like.
[0386] It will be appreciated that structure models are preferably
generated by a computing platform, which generates a graphic output
of the models via a display generating device such as screen or
printer. The computing platform generates graphic representations
of atomic structure models via a processing unit which processes
structure coordinate data stored in a retrievable format in the
data storage device.
[0387] Suitable software applications, well known to those of skill
in the art, which may be used by the processing unit to process
structure coordinate data so as to provide a graphic output of
three-dimensional structure models generated therewith via display
include, for example, RIBBONS (Carson, M., 1997. Methods in
Enzymology 277, 25), 0 (Jones, T A. et al., 1991. Acta Crystallogr
A47, 110), DINO (DINO: Visualizing Structural Biology (2001)
wwwdotdino3ddotorg); and QUANTA, CHARMM, INSIGHT, SYBYL, MACROMODE,
ICM, MOLMOL, RASMOL and GRASP (reviewed in Kraulis, J., 1991. Appl
Crystallogr. 24, 946).
As mentioned hereinabove, once a structural model of GSK-1 is
obtained substances which specifically bind the Phe93 residue in
the active site of the model are identifiable. This is preferably
effected using Rational Drug Design (RDD).
[0388] One approach to identify a putative inhibitor via rational
drug design is by screening a chemical and/or peptide structure
database ("3D database"), using software employing "scanner" type
algorithms. Such software applications utilize atomic coordinates
defining the three-dimensional structure of a binding pocket of a
molecule and of a chemical structure stored in the database to
computationally model the "docking" of the screened chemical
structure with the binding pocket so as to qualify the binding of
the binding pocket, or of the indicated amino acid therein, with
the chemical structure. Iterating this process with each of a
plurality of chemical structures stored in the database therefore
enables computational screening of such a plurality to identify a
chemical structure potentially having a desired binding interaction
with the binding pocket, or with the indicated amino acid residue
therein, and hence the putative inhibitor.
[0389] Any commercially available library of chemical structures of
small molecules and/or peptides can be used as a suitable chemical
structure database for identifying the inhibitor as described
herein.
[0390] Alternatively, identifying the inhibitor can be effected
using de novo rational drug design, or via modification of a known
chemical structure. In such case, software comprising "builder"
type algorithms utilizes a set of atomic coordinates defining a
three-dimensional structure of the binding pocket and the
three-dimensional structures of basic chemical building blocks to
computationally assemble a putative inhibitor. Such an approach may
be employed to structurally refine a putative inhibitor identified,
for example, via chemical database screening as described
above.
[0391] Ample guidance for performing rational drug design by
utilizing software employing such "scanner" and "builder" type
algorithms is available in the literature.
[0392] Criteria employed by software programs used in rational drug
design to qualify the binding of screened chemical structures with
binding pockets include gap space, hydrogen bonding, electrostatic
interactions, van der Waals forces, hydrophilicity/hydrophobicity,
etc. Generally, the greater the contact area between the screened
substance and the indicated binding site of the enzyme, the lower
the steric hindrance, the lower the "gap space", and the greater
the number of at least the hydrophobic interactions, the greater
will be the capacity of the screened substance to bind to the
indicated amino acid residue within the binding site of GSK-3.
[0393] The "gap space" refers to unoccupied space between the van
der Waals surface of a screened substance positioned within a
binding pocket and the surface of the binding pocket defined by
amino acid residues in the binding pocket. Gap space may be
identified, for example, using an algorithm based on a series of
cubic grids surrounding the docked molecule.
[0394] Modeling or docking may be followed by energy minimization
with standard molecular mechanics force fields or dynamics with
programs known in the art.
[0395] In some embodiments, once a putative substance is identified
in silico, it is further tested by "wet" experiments, by
determining, in vitro, an inhibition of an activity of GSK-3 by the
substance, as described herein.
[0396] In some embodiments, in order to further substantiate that
the substance is an effective substrate-competitive inhibitor, its
binding to Phe93 is determined by comparing an inhibition of an
activity of a wild-type GSK-3 to an inhibition of an activity of a
mutated GSK-3 that comprises an amino acid substitution with
respect to Phe93 (e.g., as set forth in SEQ ID NO:9), as described
herein.
[0397] Thus, a substance that (i) is in silico identified suitable
to bind Phe93 in a GSK-3 enzyme; (ii) inhibits an activity of GSK-3
in in vitro assays for determining a kinase activity in the
presence and absence of the substance; and (iii) inhibits an
activity of a Phe93-mutated GSK-3 enzyme by less than 20%, is
identified as a putative (potent) GSK-3 substrate competitive
inhibitor.
[0398] General:
[0399] The terms "comprises", "comprising", "includes",
"including", "having" and their conjugates mean "including but not
limited to".
[0400] The term "consisting of" means "including and limited
to".
[0401] The term "consisting essentially of" means that the
composition, method or structure may include additional
ingredients, steps and/or parts, but only if the additional
ingredients, steps and/or parts do not materially alter the basic
and novel characteristics of the claimed composition, method or
structure.
[0402] The word "exemplary" is used herein to mean "serving as an
example, instance or illustration". Any embodiment described as
"exemplary" is not necessarily to be construed as preferred or
advantageous over other embodiments and/or to exclude the
incorporation of features from other embodiments.
[0403] The word "optionally" is used herein to mean "is provided in
some embodiments and not provided in other embodiments". Any
particular embodiment of the invention may include a plurality of
"optional" features unless such features conflict.
[0404] As used herein, the singular form "a", "an" and "the"
include plural references unless the context clearly dictates
otherwise. For example, the term "a compound" or "at least one
compound" may include a plurality of compounds, including mixtures
thereof.
[0405] Throughout this application, various embodiments of this
invention may be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed subranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as
well as individual numbers within that range, for example, 1, 2, 3,
4, 5, and 6. This applies regardless of the breadth of the
range.
[0406] Whenever a numerical range is indicated herein, it is meant
to include any cited numeral (fractional or integral) within the
indicated range. The phrases "ranging/ranges between" a first
indicate number and a second indicate number and "ranging/ranges
from" a first indicate number "to" a second indicate number are
used herein interchangeably and are meant to include the first and
second indicated numbers and all the fractional and integral
numerals therebetween.
[0407] As used herein the term "method" refers to manners, means,
techniques and procedures for accomplishing a given task including,
but not limited to, those manners, means, techniques and procedures
either known to, or readily developed from known manners, means,
techniques and procedures by practitioners of the chemical,
pharmacological, biological, biochemical and medical arts.
[0408] As used herein, the terms "polypeptide" and "peptide"
encompass an amino acid sequence of any length including
full-length proteins or portions thereof, wherein the amino acid
residues are linked by covalent peptide bonds. Generally, an amino
acid sequence of 50 amino acids and more are referred to herein as
"polypeptide" or "protein", and an amino acid sequence of less than
50 amino acids is referred to herein as "peptide".
[0409] The term "peptide" as used herein encompasses also peptoids
and semipeptoids which are peptide analogs, which may have, for
example, modifications rendering the peptides more stable while in
a body or more capable of penetrating into cells. Such
modifications include, but are not limited to N-terminus
modification, C-terminus modification, peptide bond modification,
including, but not limited to, CH.sub.2--NH, CH.sub.2--S,
CH.sub.2--S.dbd.O, O.dbd.C.ident.NH, CH.sub.2--O,
CH.sub.2--CH.sub.2, S.dbd.C.ident.NH, CH.dbd.CH or CF.dbd.CH,
backbone modifications, and residue modification. Methods for
preparing peptidomimetic compounds are well known in the art and
are specified, for example, in Quantitative Drug Design, C. A.
Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which
is incorporated by reference as if fully set forth herein. Further
details in this respect are provided hereinunder.
[0410] In some embodiments, the peptides described herein are
chemically synthesized peptides.
[0411] Peptide bonds (--CO--NH--) within the peptide may be
substituted, for example, by N-methylated bonds
(--N(CH.sub.3)--CO--), ester bonds (--C(R)H--C--O--O--C(R)--N--),
ketomethylen bonds (--CO--CH.sub.2--), .alpha.-aza bonds
(--NH--N(R)--CO--), wherein R is any alkyl, e.g., methyl, carba
bonds (--CH.sub.2--NH--), hydroxyethylene bonds
(--CH(OH)--CH.sub.2--), thioamide bonds (--CS--NH--), olefinic
double bonds (--CH.dbd.CH--), retro amide bonds (--NH--CO--),
peptide derivatives (--N(R)--CH.sub.2--CO--), wherein R is the
"normal" side chain, naturally presented on the carbon atom.
[0412] These modifications can occur at any of the bonds along the
peptide chain and even at several (2-3) at the same time.
[0413] As used herein, the phrase "amino acid residue", which is
also referred to herein, interchangeably, as "amino acid",
describes an amino acid unit within a polypeptide chain. The amino
acid residues within the peptides described herein can be either
natural or modified amino acid residues, as these phrases are
defined hereinafter.
[0414] As used herein, the phrase "natural amino acid residue"
describes an amino acid residue, as this term is defined
hereinabove, which includes one of the twenty amino acids found in
nature.
[0415] As used herein, the phrase "modified amino acid residue"
describes an amino acid residue, as this term is defined
hereinabove, which includes a natural amino acid that was subjected
to a modification at its side chain. Such modifications are well
known in the art and include, for example, incorporation of a
functionality group such as, but not limited to, a hydroxy group,
an amino group, a carboxy group and a phosphate group within the
side chain. This phrase therefore includes, unless otherwise
specifically indicated, chemically modified amino acids, including
amino acid analogs (such as penicillamine, 3-mercapto-D-valine),
naturally-occurring non-proteogenic amino acids (such as
norleucine), and chemically-synthesized compounds that have
properties known in the art to be characteristic of an amino acid.
The term "proteogenic" indicates that the amino acid can be
incorporated into a protein in a cell through well-known metabolic
pathways.
[0416] Accordingly, as used herein, the term "amino acid" or "amino
acids" is understood to include the 20 naturally occurring amino
acids; those amino acids often modified post-translationally in
vivo, including, for example, hydroxyproline, phosphoserine and
phosphothreonine; and other unusual amino acids including, but not
limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine,
nor-valine, nor-leucine and ornithine. Furthermore, the term "amino
acid" includes both D- and L-amino acids which are linked via a
peptide bond or a peptide bond analog to at least one addition
amino acid as this term is defined herein.
[0417] The peptides of the present embodiments are preferably
utilized in a linear form, although it will be appreciated that in
cases where cyclization does not severely interfere with peptide
characteristics, cyclic forms of the peptide can also be
utilized).
[0418] Cyclic peptides can either be synthesized in a cyclic form
or configured so as to assume a cyclic form under desired
conditions (e.g., physiological conditions).
[0419] The peptides of the present embodiments are preferably
peptidomimetics, as this term is define hereinabove, which mimic
the structural features of the critical amino acid motif
ZX.sub.1X.sub.2X.sub.3S(p), as is further detailed hereinabove.
[0420] Protein phosphorylation plays a crucial part in the
biochemical control of cellular activity. Phosphorylation usually
means formation of a phosphate ester bond between a phosphate
(PO.sub.4) group and an amino acid containing a hydroxyl (OH) group
(tyrosine, serine and threonine). Many phosphorylation sites in
proteins act as recognition elements for binding to other proteins,
and those binding events activate or deactivate signaling and other
pathways. Protein phosphorylation thus acts as a switch to turn
biochemical signaling on and off.
[0421] Phosphopeptide mimetics are a subclass of peptidomimetics
that contain analogs of phosphorylated tyrosine, serine and
threonine. Phosphate esters may be hydrolyzed by various enzymes,
thus turning off a phosphorylation signal. Phosphopeptide mimetics,
however, usually contain non-hydrolyzable analogs to prevent
inactivation (Burke et al, 1994a; Burke et al, 1996a; Chen et al,
1995; Wiemann et al, 2000; Shapiro et al, 1997; Otaka et al, 1995;
Otaka et al, 2000). General examples of phosphopeptide mimetics in
the art include SH2 domain analogs (Burke et al, 1994a; Fu et al,
1998; Gao et al, 2000; Mikol et al, 1995; Ye et al, 1995),
transcription factor NF-(kappa)B analog (McKinsey et al, 1997), P53
analog (Higashimoto et al, 2000) and protein-tyrosine phosphatase
inhibitors (Burke et al, 1994b; Burke et al, 1996b; Groves et al,
1998; Kole et al, 1995; Kole et al, 1997; Roller et al, 1998).
[0422] Commercially available software packages can be used to
design small peptides and/or peptidomimetics containing,
phosphoserine or phosphothreonine analogs, preferably
non-hydrolyzable analogs, as specific antagonists/inhibitors.
Suitable commercially available software for analyzing crystal
structure, designing and optimizing small peptides and
peptidomimetics include, but are not limited to: Macromolecular
X-ray Crystallography QUANTA Environment (Molecular Simulations,
Inc.); TeXsan, BioteX, and SQUASH (Molecular Structure
Corporation); and Crystallographica (Oxford Cryostsystems).
[0423] The peptides according to the present embodiments can
further include salts and chemical derivatives of the peptides. As
used herein, the phrase "chemical derivative" describes a peptide
as described herein having one or more residues chemically
derivatized by reaction of a functional side group. Such
derivatized molecules include, for example, those molecules in
which free amino groups have been derivatized to form amine
hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,
t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups.
Free carboxyl groups may be derivatized to form salts, methyl and
ethyl esters or other types of esters or hydrazides. Free hydroxyl
groups may be derivatized to form O-acyl or O-alkyl derivatives.
Also included as chemical derivatives are those peptides that
contain one or more naturally occurring amino acid derivatives of
the twenty standard amino acids. For example, 4-hydroxyproline may
be substituted for proline; 5-hydroxylysine may be substituted for
lysine; 3-methylhistidine may be substituted for histidine;
homoserine may be substituted for serine; and ornithine may be
substituted for lysine. The chemical derivatization does not
comprehend changes in functional groups which change one amino acid
to another.
[0424] As is mentioned hereinabove, some useful modifications are
designed to increase the stability of the peptide in solution and,
therefore, serve to prolong the half-life of the peptide in
solutions, particularly biological fluids, such as blood, plasma or
serum, by blocking proteolytic activity in the blood. Hence, the
peptides described herein can have a stabilizing group at one or
both termini. Typical stabilizing groups include amido, acetyl,
benzyl, phenyl, tosyl, alkoxycarbonyl, alkyl carbonyl,
benzyloxycarbonyl and the like end group modifications. Additional
modifications include using one or more "D" amino acids in place of
"L" amino acid(s), cyclization of the peptide inhibitor, and amide
rather than amino or carboxy termini to inhibit exopeptidase
activity.
[0425] The peptides described herein may or may not be
glycosylated. The peptides are not glycosylated, for example, when
produced directly by peptide synthesis techniques or are produced
in a prokaryotic cell transformed with a recombinant
polynucleotide. Eukaryotically-produced peptide molecules are
typically glycosylated. The term "hydrocarbon", as used herein,
encompasses any moiety that is based on a linear and/or cyclic
chain of carbons which are mainly substituted by hydrogens. A
hydrocarbon can be a saturated or unsaturated moiety, and can
optionally be substituted by one or more substituents, as described
herein.
[0426] The term "alkyl" describes a saturated aliphatic hydrocarbon
including straight chain and branched chain groups. Preferably, the
alkyl group has 1 to 20 carbon atoms. Whenever a numerical range;
e.g., "1-20", is stated herein, it implies that the group, in this
case the alkyl group, may contain 1 carbon atom, 2 carbon atoms, 3
carbon atoms, etc., up to and including 20 carbon atoms. More
preferably, the alkyl is a medium size alkyl having 2 to 10 carbon
atoms. Most preferably, unless otherwise indicated, the alkyl is a
lower alkyl having 2 to 6 carbon atoms. The alkyl group may be
substituted or unsubstituted, as defined herein.
[0427] The term "cycloalkyl" describes an all-carbon monocyclic or
fused ring (i.e., rings which share an adjacent pair of carbon
atoms) group where one or more of the rings does not have a
completely conjugated pi-electron system. The cycloalkyl group may
be substituted or unsubstituted.
[0428] The term "heteroalicyclic" describes a monocyclic or fused
ring group having in the ring(s) one or more atoms such as
nitrogen, oxygen and sulfur. The rings may also have one or more
double bonds. However, the rings do not have a completely
conjugated pi-electron system. The heteroalicyclic may be
substituted or unsubstituted. Representative examples are
piperidine, piperazine, tetrahydrofurane, tetrahydropyrane,
morpholino and the like.
[0429] The term "aryl" describes an all-carbon monocyclic or
fused-ring polycyclic (i.e., rings which share adjacent pairs of
carbon atoms) groups having a completely conjugated pi-electron
system. The aryl group may be substituted or unsubstituted.
[0430] The term "heteroaryl" describes a monocyclic or fused ring
(i.e., rings which share an adjacent pair of atoms) group having in
the ring(s) one or more atoms, such as, for example, nitrogen,
oxygen and sulfur and, in addition, having a completely conjugated
pi-electron system. Examples, without limitation, of heteroaryl
groups include pyrrole, furane, thiophene, imidazole, oxazole,
thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline
and purine. The heteroaryl group may be substituted or
unsubstituted.
[0431] Whenever an alkyl, cycloalkyl, heteroalicyclic, aryl,
heteroaryl or a hydrocarbon is substituted by one or more
substituents, each substituent group can independently be, for
example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl,
aryl, heteroaryl, heteroalicyclic, amine, halide, sulfonate,
sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy,
thioalkoxy, thioaryloxy, cyano, nitro, azo, sulfonamide,
C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate,
urea, thiourea, N-carbamate, O-carbamate, C-amide, N-amide, guanyl,
guanidine and hydrazine.
[0432] A "hydroxy" group refers to an --OH group.
[0433] An "azide" group refers to a --N.dbd.N.sup.+.dbd.N.sup.-
group.
[0434] An "alkoxy" group refers to both an --O-alkyl and an
--O-cycloalkyl group, as defined herein.
[0435] An "aryloxy" group refers to both an --O-aryl and an
--O-heteroaryl group, as defined herein.
[0436] A "thiohydroxy" or "thiol" group refers to a --SH group.
[0437] A "thioalkoxy" group refers to both an --S-alkyl group, and
an --S-cycloalkyl group, as defined herein.
[0438] A "thioaryloxy" group refers to both an --S-aryl and an
--S-heteroaryl group, as defined herein.
[0439] A "carbonyl" group refers to a --C(.dbd.O)--R' group, where
R' is defined as hereinabove.
[0440] A "thiocarbonyl" group refers to a --C(.dbd.S)--R' group,
where R' is as defined herein.
[0441] A "C-carboxy" group refers to a --C(.dbd.O)--O--R' groups,
where R' is as defined herein.
[0442] An "O-carboxy" group refers to an R'C(.dbd.O)--O-- group,
where R' is as defined herein.
[0443] An "oxo" group refers to a=O group.
[0444] A "carboxylate" or "carboxyl" encompasses both C-carboxy and
O-carboxy groups, as defined herein.
[0445] A "carboxylic acid" group refers to a C-carboxy group in
which R' is hydrogen.
[0446] A "thiocarboxy" or "thiocarboxylate" group refers to both
--C(.dbd.S)--O--R' and --O--C(.dbd.S)R' groups.
[0447] An "ester" refers to a C-carboxy group wherein R' is not
hydrogen.
[0448] An ester bond refers to a --O--C(.dbd.O)-- bond.
[0449] A "halo" group refers to fluorine, chlorine, bromine or
iodine.
[0450] A "sulfinyl" group refers to an --S(.dbd.O)--R' group, where
R' is as defined herein.
[0451] A "sulfonyl" group refers to an --S(.dbd.O).sub.2--R' group,
where R' is as defined herein.
[0452] A "sulfonate" group refers to an --S(.dbd.O).sub.2--O--R'
group, where R' is as defined herein.
[0453] A "sulfate" group refers to an --O--S(.dbd.O).sub.2--O--R'
group, where R' is as defined as herein.
[0454] A "sulfonamide" or "sulfonamido" group encompasses both
S-sulfonamido and N-sulfonamido groups, as defined herein.
[0455] An "S-sulfonamido" group refers to a
--S(.dbd.O).sub.2--NR'R'' group, with each of R' and R'' as defined
herein.
[0456] An "N-sulfonamido" group refers to an
R'S(.dbd.O).sub.2--NR'' group, where each of R' and R'' is as
defined herein.
[0457] An "O-carbamyl" group refers to an --OC(.dbd.O)--NR'R''
group, where each of R' and R'' is as defined herein.
[0458] An "N-carbamyl" group refers to an R'OC(.dbd.O)--NR''--
group, where each of R' and R'' is as defined herein.
[0459] A "carbamyl" or "carbamate" group encompasses O-carbamyl and
N-carbamyl groups.
[0460] A carbamate bond describes a --O--C(.dbd.O)--NR'-- bond,
where R' is as described herein.
[0461] An "O-thiocarbamyl" group refers to an --OC(.dbd.S)--NR'R''
group, where each of R' and R'' is as defined herein.
[0462] An "N-thiocarbamyl" group refers to an R'OC(.dbd.S)NR''--
group, where each of R' and R'' is as defined herein.
[0463] A "thiocarbamyl" or "thiocarbamate" group encompasses
O-thiocarbamyl and N-thiocarbamyl groups.
[0464] A thiocarbamate bond describes a --O--C(.dbd.S)--NR'-- bond,
where R' is as described herein.
[0465] A "C-amido" group refers to a --C(.dbd.O)--NR'R'' group,
where each of R' and R'' is as defined herein.
[0466] An "N-amido" group refers to an R'C(.dbd.O)--NR''-- group,
where each of R' and R'' is as defined herein.
[0467] An "amide" group encompasses both C-amido and N-amido
groups.
[0468] An amide bond describes a --NR'--C(.dbd.O)-- bond, where R'
is as defined herein.
[0469] A "urea" group refers to an --N(R')--C(.dbd.O)--NR''R'''
group, where each of R' and R'' is as defined herein, and R''' is
defined as R' and R'' are defined herein.
[0470] A "nitro" group refers to an --NO.sub.2 group.
[0471] A "cyano" group refers to a --C.ident.N group.
[0472] The term "phosphonyl" or "phosphonate" describes a
--P(.dbd.O)(OR')(OR'') group, with R' and R'' as defined
hereinabove.
[0473] The term "phosphate" describes an --O--P(.dbd.O)(OR')(OR'')
group, with each of R' and R'' as defined hereinabove.
[0474] A "phosphoric acid" is a phosphate group is which each of R
is hydrogen.
[0475] The term "phosphinyl" describes a --PR'R'' group, with each
of R' and R'' as defined hereinabove.
[0476] The term "thiourea" describes a --N(R')--C(.dbd.S)--NR''--
group, with each of R' and R'' as defined hereinabove.
[0477] Any of the substances described herein (e.g., peptides,
polypeptides or small molecules), can be in a form of a
pharmaceutically acceptable salt thereof.
[0478] The phrase "pharmaceutically acceptable salt" refers to a
charged species of the parent compound and its counter ion, which
is typically used to modify the solubility characteristics of the
parent compound and/or to reduce any significant irritation to an
organism by the parent compound, while not abrogating the
biological activity and properties of the administered
compound.
[0479] The present invention further encompasses prodrugs, solvates
and hydrates of the substances described herein.
[0480] As used herein, the term "prodrug" refers to an agent, which
is converted into the active compound (the active parent drug) in
vivo. Prodrugs are typically useful for facilitating the
administration of the parent drug. They may, for instance, be
bioavailable by oral administration whereas the parent drug is not.
The prodrug may also have improved solubility as compared with the
parent drug in pharmaceutical compositions. Prodrugs are also often
used to achieve a sustained release of the active compound in vivo.
An example, without limitation, of a prodrug would be a peptide, as
described herein, having one or more carboxylic acid moieties,
which is administered as an ester (the "prodrug"). Such a prodrug
is hydrolysed in vivo, to thereby provide the free compound (the
parent drug). The selected ester may affect both the solubility
characteristics and the hydrolysis rate of the prodrug.
[0481] The term "solvate" refers to a complex of variable
stoichiometry (e.g., di-, tri-, tetra-, penta-, hexa-, and so on),
which is formed by a solute (the peptide) and a solvent, whereby
the solvent does not interfere with the biological activity of the
solute. Suitable solvents include, for example, ethanol, acetic
acid and the like.
[0482] The term "hydrate" refers to a solvate, as defined
hereinabove, where the solvent is water.
[0483] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable subcombination
or as suitable in any other described embodiment of the invention.
Certain features described in the context of various embodiments
are not to be considered essential features of those embodiments,
unless the embodiment is inoperative without those elements.
[0484] Various embodiments and aspects of the present invention as
delineated hereinabove and as claimed in the claims section below
find experimental support in the following examples.
EXAMPLES
[0485] Reference is now made to the following examples, which
together with the above descriptions illustrate some embodiments of
the invention in a non limiting fashion.
Material and Methods
[0486] Materials:
[0487] Peptides were synthesized by Genemed Synthesis, Inc. (San
Francisco, USA). The peptide substrates included p9CREB,
ILSRRPS(p)YR (SEQ ID NO:18); pIRS-1, RREGGMSRPAS(p)VDG (SEQ ID
NO:19); and PGS-1, YRRAAVPPSPSLSRHSSPSQS(p)EDEEE (SEQ ID NO:20) as
previously described [Ilouz et al. (2006) supra].
[0488] Other L803 variants were synthesized as described
herein.
[0489] Anti GSK-3.beta. antibody was obtained from Transduction
Laboratory (Lexington, Ky., USA).
[0490] Anti-phospho-GSK3 (Y.sup.216) was obtained from Upstate
Biotechnology (Lake Placid, N.Y., USA).
[0491] Anti-phospho-CREB (S.sup.129/133) was obtained from
BioSource International, Inc. (Camarillo, Calif., USA).
[0492] CREB antibody was from Cell Signaling Technology (Beverly,
Mass., USA).
[0493] Anti-phospho-IRS-1 (5.sup.332) was generated as previously
described [Liberman, Z. & Eldar-Finkelman, H. (2005)
supra].
[0494] Radioactive materials were purchased from NEN PerkinElmer
USA.
[0495] Plasmids and Mutants:
[0496] GSK-3.beta. in the pCMV4 vector was used as the template for
mutagenesis. Mutations were generated using QuickChange Site
Directed Mutagenesis Kit (Stratagene, La Jolla, Calif., USA)
according the manufacturer's protocols. Mutations included
replacement of D90, K91, R92, F93, K94, V214 to alanine, F93 to
tyrosine, and a triple mutation at residues 91-93. All constructs
were sequenced to confirm the presence of desired mutations. The
sequences of mutagenic oligonucleotides are available from the
inventors upon request.
[0497] N'IRS-1 (also termed PTB2) plasmid was previously described
[[Liberman, Z. & Eldar-Finkelman, H. (2005) supra].
[0498] CREB-GFP plasmid was purchased from Clontech (Mountain View,
Calif., USA).
[0499] Cell Transfections and Protein Partial Purifications:
[0500] HEK-293 cells were grown in Dulbecco's Modified Eagle's
Medium (DMEM), supplemented with 10% fetal calf serum (FCS), 2 mM
glutamine, 100 units/ml penicillin, and 100 .mu.g/ml streptomycin.
HEK-293 cells were transiently transfected with indicated
constructs, using calcium phosphate method as described [Ilouz et
al. (2006) supra]. Cells were lysed in ice-cold buffer G (20 mM
Tris-HCl, pH 7.5, 10 mM .beta.-glycerophosphate, 10% glycerol, 1 mM
EGTA, 1 mM EDTA, 50 mM NaF, 5 mM sodium pyrophosphate, 0.5 mM
orthovanadate, 1 mM benzamidine, 10 .mu.g/ml leupeptin, 5 .mu.g/ml
aprotinin, 1 .mu.g/ml pepstatin, and 0.5% Triton X100). Cell
extracts were centrifuged for 30 minutes at 15,000.times.g.
Supernatants were collected, and equal amounts of proteins were
boiled with SDS sample buffer and subjected to gel electrophoresis
(7.5-12% polyacrylamide gel), transferred to nitrocellulose
membranes, and immunoblotted with indicated antibodies. For partial
purification, cells were lysed in buffer H (20 mM Tris, pH 7.3, 1
mM EGTA, 1 mM EDTA, 1 mM orthovanadate, 25 .mu.g/ml leupeptin, 25
.mu.g/ml aprotinin, and 25 .mu.g/ml pepstatin A, 500 nM
microcystine LR, and 0.25% Triton X100). The lysates were
centrifuged at 15,000.times.g. The resulting supernatants were
passed through DE-52 (Whatman, Maidstone, England) mini-columns
that were equilibrated with buffer H. GSK-3.beta. proteins were
eluted with the same buffer containing 0.02 M NaCl. Equal amounts
of proteins were used for in vitro kinase assays. In all
experiments, GSK-3 mutants were expressed at levels at least 5-fold
higher than levels of the endogenous GSK-3.beta. as determined by
western blot analysis.
[0501] In Vitro Kinase Assays:
[0502] The GSK-3.beta. proteins (WT or mutants) were incubated with
indicated substrate in a reaction mixture (50 mM Tris-HCl, pH 7.3,
10 mM magnesium acetate, and 0.01% .beta.-mercaptoethanol) together
with 100 .mu.M .sup.32P[.gamma.-ATP] (0.5 .mu.Ci/assay) for 15
minutes. Reactions were stopped by spotting on p81 paper (Whatman)
washed with phosphoric acid, and counted for radioactivity as
previously described [Ilouz et al. (2006) supra]. In assays from
cells overexpressing GSK-3 proteins, the activity of the endogenous
GSK-3 that was determined in cells transfected with the pCMV4
vector was subtracted from the activity values obtained for WT and
mutants.
[0503] Statistical Analysis:
[0504] Data were analyzed with Origin Professional 6.0 software
using Student's t-test to compare GSK-3 activity of WT vs mutants
or peptides-treatment vs. non treatment. Data were considered
significant at p<0.05
[0505] Molecular Dynamics:
[0506] Molecular dynamics (MD) simulations were performed with the
program Gromacs [Van Der Spoel et al. (2005) J. Comput. Chem. 26,
1701-18] employing the united atoms gromos96 43a1 force field [van
Gunsteren et al. (1996). Biomolecular Simulation: The Gromos 96
Manual and User Guide] modified to include phosphorylated residues
[wwwdotgromacsdotorg/Downloads/User_contributions/Force_fields].
[0507] The initial model of the solute (peptide or
GSK-3.beta./peptide complex) was immersed in a cube of water,
neutralized and energy minimized. This was followed by a lns MD
simulation to equilibrate the water, keeping the non hydrogen atoms
of the solute restrained. Next, additional 1 or 2 ns simulation was
performed, of the peptide or GSK-3.beta./peptide complex in water.
In the latter case the Ca atoms of GSK-3.beta. were restrained and
weak restrains were imposed on the distances between the phosphate
oxygens of S10(p) in the peptide and the side chain nitrogen atoms
of GSK-3.beta. Arg 96, Arg 180, and Lys 205. Only the last lns of
each MD simulation was considered in the analysis of the trajectory
(0.5 ns for the free peptides).
[0508] Rigid Body Docking:
[0509] Rigid body docking was performed with the
geometric-electrostatic-hydrophobic version of MolFit [Berchanski
et al. (2004) Proteins 351, 309-26]. The starting geometry of the
GSK-3.beta./ATP complex was modeled as described before [Ilouz et
al. (2006) supra], the starting geometry of the free peptide was
the representative conformer of the largest cluster obtained in MD
simulation of the free peptide in water. The comprehensive docking
scan was followed by a new post-scan filtering procedure that
incorporates statistical propensity measures and desolvation energy
calculations [Kowalsman & Eisenstein (2009) Proteins 77,
297-318]. The filtered models were further screened requesting that
S10(p) of the peptide makes contact with the positive cavity on the
surface of GSK-3.beta..
[0510] Anchoring Spots Mapping:
[0511] Anchoring spots mapping identifies preferred binding
positions of amino acid side chains on the surface of a protein
[Ben-Shimon and Eisenstein (2010) J. Mol. Biol. 402, 259-77]. This
procedure was used here to detect amino acids that bind in the
GSK-3.beta. surface cavity bordered by loop 89-95 and the P-loop.
Only side chains that bind with .DELTA.G.ltoreq.3 Kcal/mol were
considered.
Example 1
Defining a Substrate Binding Subsite in GSK-3
[0512] The Q89-N95 Segment:
[0513] The sequence segment delimited by Gln 89 and Asn 95 (see,
SEQ ID NO:2), two residues that were found to participate in GSK-3
substrate binding [Ilouz et al. 2006, supra], forms a loop (termed
herein 89-95 loop) that together with the conserved P-loop, defines
the borders of a surface cavity.
[0514] To further explore the role of the 89-95 loop in GSK-3.beta.
substrate binding, each of the amino acid residues within this
segment was individually mutated to alanine (see, FIG. 1A). HEK-293
cells were transiently transfected with cDNA constructs expressing
wild-type (WT) GSK-3.beta. (SEQ ID NO:1), D90A (SEQ ID NO:6), K91A
(SEQ ID NO:7), R92A (SEQ ID NO:8), F93A (SEQ ID NO:9), R94A (SEQ ID
NO:10) mutant proteins, as described in the Methods section
hereinabove. Cell extracts were subjected to western blot analysis
using either anti-GSK-3.beta. or antiphospho-GSK-3 (Tyr 216/Tyr 274
for .alpha. or .beta. isoforms respectively) antibodies. Control
(C) represents extracts from cells expressing the empty vector.
[0515] All the mutants were expressed at levels considerably above
that of the endogenous GSK-3.beta. (FIG. 1B, upper panel). Like the
wild-type (WT) GSK-3.beta., the mutants were phosphorylated at Tyr
216 (FIG. 1B, lower panel), indicating that their catalytic
activity was not impaired by the mutation, as phosphorylation at
Tyr 216 reflects an auto-phosphorylation process [as previously
described in Cole et al. (2004) Biochem. J. 377, 249-55; and
Eldar-Finkelman et al. (1996) Proc. Natl. Acad. Sci. USA 93,
10228-10233].
[0516] The GSK-3.beta. mutants were partially purified by ion
exchange chromatography, and their abilities to phosphorylate
peptide substrates were tested in in vitro kinase assays. The
substrates were: pIRS-1, p9CREB, and pGS-1, phosphorylated peptides
derived from the insulin receptor substrate-1 (IRS-1), cAMP
responsive element binding protein (CREB), and glycogen synthase,
respectively.
[0517] The results are presented in Table 1, as the percentage of
substrate phosphorylation (indicated peptides, pIRS-1, p9CREB and
pGS-1) obtained with WTGSK-3.beta. which was set to 100%, and are
mean of 2-3 independent experiments each performed in
duplicates.+-.SEM.
[0518] As shown in Table 1, three of the five mutants, R92A, F93A,
K94A mutants, impaired the ability to phosphorylate the substrates;
that Mutation at Lys 91 enhanced substrate phosphorylation by about
20-30%; and, notably, that mutation at Phe 93 had the most
deleterious effect for all substrates, reducing the kinase ability
to phosphorylate them by more than 50% (see also FIG. 1C). A
similar impact was observed with Q89A and N95A mutants [see, Ilouz
et al., 2006, supra].
TABLE-US-00001 TABLE 1 Substrate phosphorylation (% of
WTGSK-3.beta.) Mutant pIRS-1 p9CREB pGS-1 D90A (SEQ ID 88 .+-. 15
92 .+-. 21 83 .+-. 2 NO: 6) K91A (SEQ ID 140 .+-. 18 161 .+-. 5 119
.+-. 5 NO: 7) R92A (SEQ ID 60 .+-. 3 49 .+-. 19 41 .+-. 14 NO: 8)
F93A (SEQ ID NO: 9) 42 .+-. 13 46 .+-. 2 13 .+-. 7 K94A (SEQ ID 52
.+-. 19 71 .+-. 4 19 .+-. 14 NO: 10)
[0519] FIG. 1C presents the phosphorylation of peptide substrates
by F93A mutant. F93A was subjected to in vitro kinase assays with
substrates pIRS-1, p9CREB, and PGS-1 as described in the Methods
section hereinabove. The percentage of substrate phosphorylation
obtained with WT-GSK-3.beta. was defined as 100%, and results are
means of 2-3 independent experiments each performed in
duplicates.+-.SEM.
[0520] Hence, Phe 93 adjoins Gln 89 and Asn 95 as an important
substrate binding position. Phe 93 is located at the center of the
89-95 loop, it is highly exposed (81% solvent accessibility) and it
faces the substrate binding subsite, facilitating contacts with
variety of residues.
[0521] The role of Phe 93 in substrate binding by employing a
cellular system and protein substrates (i.e., not peptides) was
further explored. To this end, the WT-GSK-3.beta. and F93A mutant
were expressed in HEK-293 cells together with GSK-3 substrates CREB
or N'IRS-1 (the N-terminal region of IRS-1). Because GSK-3 requires
pre-phosphorylation of its substrates, the cells were treated with
forskolin to enhance CREB phosphorylation via activation of cAMP
dependent kinase (PKA), or with phorbol ester (PMA) to enhance
N'IRS-1 phosphorylation via activation of protein kinase C (PKC).
The phosphorylation of CREB at serine 129, and N'IRS-1 at serine
332 (both GSK-3 phosphorylation sites) was then examined.
[0522] Thus, HEK293 cells were co-transfected with WT-GSK-3.beta.
or F93A plasmids together with construct coding for CREB. Cells
were treated with forskolin (10 .mu.M, 1 hour), and cell extracts
were subjected to western blot analysis using anti-phospho CREB
(Ser 129/133) antibody, as presented in FIG. 1D. Expression levels
of CREB and GSK-3 proteins are indicated. The ratio of pCREB/CREB
as calculated from densitometry analysis is shown in FIG. 1E.
[0523] Similar assay was conducted using N'IRS-1 cDNA construct
instead of CREB, and cells were treated with PMA (100 nM, 30
minutes). Anti-phospho IRS-1 (Ser 332) antibody was used as
indicated, and the results are presented in FIG. 1F. Expression
levels of N'IRS-1 and GSK-3.beta. are indicated. The ratio of
pN'IRS-1/N'IRS-1 as calculated from densitometry analysis is shown
in FIG. 1G. Results are means of three independent
experiments.+-.SEM.
[0524] Unlike WT-GSK-3.beta., expression of F93A did not enhance
the phosphorylation of these substrates as determined by specific
anti-phospho-antibodies (see, FIGS. 1D-1G). This substantiated the
in vitro results showing that Phe 93 interacts with GSK-3
substrates in cellular conditions.
[0525] The Role of Phe 93 in the Inhibition of GSK-3 by the
Substrate Competitive Inhibitor L803-Mts:
[0526] Purified GSK-3.beta. was subjected to in vitro kinase assays
using pIRS-1, p9CREB, and pGS-1 substrate in the presence or
absence of L803-mts (100 .mu.M). As shown in FIG. 2A, L803-mts (SEQ
ID NO:5) competes with various substrates, indicating that its
binding mode with GSK-3 may share similar interactions to those of
GSK-3 substrates.
[0527] The interaction of L803-mts and of L803 with the 89-95 loop
was thus examined. In vitro kinase assays were performed with
WT-GSK-3.beta. and GSK-3.beta. mutants in the presence or absence
of L803-mts (SEQ ID NO:5) or L803 (SEQ ID NO:4). The results are
presented in FIGS. 2B and 2C and present the percentages of
substrate phosphorylation obtained with the inhibitor versus
phosphorylation without the inhibitor (define as 100%), and are
means of 2-3 independent experiments.+-.SEM.
[0528] The results indicated that L803-mts did not inhibit F93A,
yet was able to inhibit all other mutants including Q89A, N95A,
R92A, K94A and F93Y (data not shown). Collectively, the results
suggest that both L803-mts and L803 and the GSK-3 substrates
interact with Phe 93, but, unlike the gsk-3 substrates, L803-mts
and L803 do not interact with other residues within the 89-95 loop,
including Gln 89 and Asn 95.
Example 2
Novel Modifications of GSK-3 Peptide Inhibitors
[0529] Replacing Polar or Charged Amino Acid Residues with
Hydrophobic Residues Increases Inhibition Activity:
[0530] In view of the fact that Gln 89 and Asn 95 did not
contribute to binding of L803 or L803-mts to the catalytic site of
GSK-3, the involvement of hydrophilic interactions in the GSK-3
binding site was tested.
[0531] L803 includes two charged amino acids Lys 1 and Glu2, and a
polar residue, Gln9 (see, SEQ ID NO:4). Thus, novel peptide
variants were synthesized, in which each of these residues, Lys 1,
Glu2 and Gln9, was individually replaced by alanine. These novel
variants are termed herein PK1A (where Lys1 was replaced by
alanine; SEQ ID NO:11)), PE2A (where Glu2 was replaced by alanine;
SEQ ID NO:12), and PQ9A (where Gln9 was replaced by alanine; SEQ ID
NO:13). These modification to the sequence of the L803 peptide are
shown in FIG. 3A, where the positions that were changed to alanine
(residues 1, 2 and 9) and the substitutions of Gln9, are marked
bold.
[0532] The ability of each peptide to inhibit GSK-3.beta. was then
determined by in vitro kinase assays as described hereinabove.
Substrate phosphorylation obtained in reaction with no inhibitor
was defined as 100% (Con), and the results presented are means of
two independent experiments each performed in duplicate.+-.SEM.
[0533] As shown in FIG. 3B, PK1A (SEQ ID NO:11) inhibited
WT-GSK-3.beta. to a similar extent as L803, but inhibition by PE2A
(SEQ ID NO:12) was slightly impaired. In contrast, PQ9A (SEQ ID
NO:13), in which Gln 9 was replaced by alanine, increased the
inhibition by about two-fold relative to L803.
[0534] To further understand the contribution of position 9 to L803
function, Gln 9 was replaced with either the charged amino acid
arginine (PQ9R; SEQ ID NO:14), or the aromatic residue tyrosine
(PQ9Y; SEQ ID NO:15). The results are presented in FIG. 3C and show
that both replacements produced non-inhibitory L803 variants.
[0535] It therefore appears that the binding of L803 to GSK-3.beta.
is mostly mediated by hydrophobic interactions.
[0536] Another variant of L803 was designed based on the
experimental results for PQ9A. Assuming that the multi proline
composition of L803 and its hydrophobic nature dominate the binding
to GSK-3.beta., Gln 9 was replaced by proline, which is a small
hydrophobic residue (PQ9P; SEQ ID NO:16). In vitro kinase assays
were performed with GSK-3.beta. in the presence of L803 or PQ9P
(200 .mu.M each) and the results are presented in FIG. 4. Substrate
phosphorylation obtained without inhibitor was defined as 100%, and
results are means of two independent experiments.+-.SEM.
[0537] Indeed, PQ9P inhibited GSK-3.beta. by about 80% more
compared to L803.
[0538] The binding of PQ9P to GSK-3 substrate binding site was
further demonstrated by MD simulation, and proved supportive to its
high binding efficacy (data not shown). MD stimulation of free PQ9P
in water showed limited mobility (RMSD<1.5 .ANG. for the last
0.3 ns of the trajectory); it also showed that the peptide adopts a
different conformation than L803 (data not shown). Simulation of
the GSK-3.beta./PQ9P complex, starting with PQ9P near the deep
groove, showed that the bound PQ9P differs considerably from the
free peptide (RMSD deviation of 5.3 .ANG. for the non hydrogen
atoms). Hence, the rigidity of this peptide does not help to lower
the entropy barrier for binding. The high affinity of PQ9P can be
attributed to the extensive contacts with Phe 93, Phe 67 and the
substrate binding subsite. PQ9P does not interact with the
hydrophobic patch formed by V214, I217 and Y216; hence its binding
resembles that of a substrate.
[0539] FIG. 5 presents comparative plots showing the inhibition
activity of L803, PQ9A and PQ9P, and clearly shows the enhanced
inhibition activity of PQ9P.
[0540] Modified Peptides Having Attached Thereto a Fatty Acid
Hydrophobic Moiety:
[0541] In order to design a cell permeable variant of PQ9P, a fatty
acid moiety was attached to its N-terminus was prepared and tested.
Myristic acid, as an exemplary fatty acid, was attached to PQ9P via
a glycine bridge. The resulting peptide was named L806-mts and had
the following amino acid sequence:
TABLE-US-00002 (SEQ ID NO: 17) Myr-GKEAPPAPPPS(p)P
[0542] In vitro kinase assays were performed with GSK-3.beta. in
the presence of L803-mts (SEQ ID NO:5) or L806-mts (SEQ ID NO:16)
at increasing concentrations and the results are presented in FIG.
6A. Substrate phosphorylation obtained without inhibitor was
defined as 100%, and results are means of two independent
experiments.+-.SEM.
[0543] Indeed, L806-mts inhibited GSK-3.beta. with IC.sub.50 of
about 1 .mu.m.
[0544] In further studies, COS-7 cells were treated with L806-mts
at various concentrations for 5 hour. Levels of .beta.-catenin were
determined by Western blot analysis using anti-.beta.-catenin
antibody, as presented in FIG. 6B. Elevation of .beta.-catenin
reflects inhibition of GSK-3.
[0545] L806-mts-treated COS-7 cells were also tested for
phosphorylation of the GSK-3 substrate heat shock factor-1 (HSF-1).
Phosphorylation of HSF-1 was determined by Western blot analysis
using anti phosphor-HSF-1 antibody, as presented in FIG. 6C.
Reduced phosphorylation proves inhibition of GSK-3.
Example 3
In Vivo Studies
[0546] C57BL/6J mice (12 week old; obtained from Animal Facilities
at Tel Aviv University) were treated with L803-mts or L806-mts via
a nasal administration (60 .mu.g peptide/per mouse/per day) for 3
days. Non-treated animals served as control. Brains were removed
and hippocampus was homogenized in ice-cold `buffer G`(20 mM Tris
pH 7.5, 10 mM .beta.-glycerophosphate, 10% glycerol, 1 mM EGTA, 1
mM EDTA, 50 mM NaF, 5 mM sodium pyrophosphate, 0.5 mM
orthovanadate, 1 mM benzamidine 5 .mu.g/ml leupeptin, 25 .mu.g/ml
aprotinin, 5 .mu.g/ml pepstatin, and 0.5% Triton X100). Equal
amounts of proteins (50 .mu.g) were subjected to gel
electrophoresis, transferred to nitrocellulose membranes, and
immunoblotted with anti-.beta.-catenin antibody. Hippocampus
.beta.-catenin levels were determined by western blot analysis as
described.
[0547] The obtained Western Blot analyses are presented in FIG. 7A
(for L803-mts and FIG. 7B (for L806-mts). Expression levels of
GSK-3.beta. are also shown. As shown in FIGS. 7A and 7B, elevation
in .beta.-catenin levels, which is indicative for in vivo
inhibition of GSK-3, was seen in the presence of L806-mts.
[0548] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims.
[0549] All publications, patents and patent applications mentioned
in this specification are herein incorporated in their entirety by
reference into the specification, to the same extent as if each
individual publication, patent or patent application was
specifically and individually indicated to be incorporated herein
by reference. In addition, citation or identification of any
reference in this application shall not be construed as an
admission that such reference is available as prior art to the
present invention. To the extent that section headings are used,
they should not be construed as necessarily limiting.
Sequence CWU 1
1
471433PRTHomo sapiens 1Met Ser Gly Arg Pro Arg Thr Thr Ser Phe Ala
Glu Ser Cys Lys Pro 1 5 10 15 Val Gln Gln Pro Ser Ala Phe Gly Ser
Met Lys Val Ser Arg Asp Lys 20 25 30 Asp Gly Ser Lys Val Thr Thr
Val Val Ala Thr Pro Gly Gln Gly Pro 35 40 45 Asp Arg Pro Gln Glu
Val Ser Tyr Thr Asp Thr Lys Val Ile Gly Asn 50 55 60 Gly Ser Phe
Gly Val Val Tyr Gln Ala Lys Leu Cys Asp Ser Gly Glu 65 70 75 80 Leu
Val Ala Ile Lys Lys Val Leu Gln Asp Lys Arg Phe Lys Asn Arg 85 90
95 Glu Leu Gln Ile Met Arg Lys Leu Asp His Cys Asn Ile Val Arg Leu
100 105 110 Arg Tyr Phe Phe Tyr Ser Ser Gly Glu Lys Lys Asp Glu Val
Tyr Leu 115 120 125 Asn Leu Val Leu Asp Tyr Val Pro Glu Thr Val Tyr
Arg Val Ala Arg 130 135 140 His Tyr Ser Arg Ala Lys Gln Thr Leu Pro
Val Ile Tyr Val Lys Leu 145 150 155 160 Tyr Met Tyr Gln Leu Phe Arg
Ser Leu Ala Tyr Ile His Ser Phe Gly 165 170 175 Ile Cys His Arg Asp
Ile Lys Pro Gln Asn Leu Leu Leu Asp Pro Asp 180 185 190 Thr Ala Val
Leu Lys Leu Cys Asp Phe Gly Ser Ala Lys Gln Leu Val 195 200 205 Arg
Gly Glu Pro Asn Val Ser Tyr Ile Cys Ser Arg Tyr Tyr Arg Ala 210 215
220 Pro Glu Leu Ile Phe Gly Ala Thr Asp Tyr Thr Ser Ser Ile Asp Val
225 230 235 240 Trp Ser Ala Gly Cys Val Leu Ala Glu Leu Leu Leu Gly
Gln Pro Ile 245 250 255 Phe Pro Gly Asp Ser Gly Val Asp Gln Leu Val
Glu Ile Ile Lys Val 260 265 270 Leu Gly Thr Pro Thr Arg Glu Gln Ile
Arg Glu Met Asn Pro Asn Tyr 275 280 285 Thr Glu Phe Lys Phe Pro Gln
Ile Lys Ala His Pro Trp Thr Lys Asp 290 295 300 Ser Ser Gly Thr Gly
His Phe Thr Ser Gly Val Arg Val Phe Arg Pro 305 310 315 320 Arg Thr
Pro Pro Glu Ala Ile Ala Leu Cys Ser Arg Leu Leu Glu Tyr 325 330 335
Thr Pro Thr Ala Arg Leu Thr Pro Leu Glu Ala Cys Ala His Ser Phe 340
345 350 Phe Asp Glu Leu Arg Asp Pro Asn Val Lys Leu Pro Asn Gly Arg
Asp 355 360 365 Thr Pro Ala Leu Phe Asn Phe Thr Thr Gln Glu Leu Ser
Ser Asn Pro 370 375 380 Pro Leu Ala Thr Ile Leu Ile Pro Pro His Ala
Arg Ile Gln Ala Ala 385 390 395 400 Ala Ser Thr Pro Thr Asn Ala Thr
Ala Ala Ser Asp Ala Asn Thr Gly 405 410 415 Asp Arg Gly Gln Thr Asn
Asn Ala Ala Ser Ala Ser Ala Ser Asn Ser 420 425 430 Thr 27PRTHomo
sapiensmisc_featureGSK-3 beta loop coresponding to amino acid
residues 89-95 2Gln Asp Lys Arg Phe Lys Asn 1 5 35PRTArtificial
sequenceGSK-3 recognition motif 3Xaa Xaa Xaa Xaa Xaa 1 5
411PRTArtificial sequenceSubstrate competitive inhibitor, L803 4Lys
Glu Ala Pro Pro Ala Pro Pro Gln Ser Pro 1 5 10 512PRTArtificial
sequenceSubstrate competitive inhibitor, L803-mts 5Gly Lys Glu Ala
Pro Pro Ala Pro Pro Gln Ser Pro 1 5 10 6433PRTArtificial
sequenceGSK-3 beta, D90A mutant 6Met Ser Gly Arg Pro Arg Thr Thr
Ser Phe Ala Glu Ser Cys Lys Pro 1 5 10 15 Val Gln Gln Pro Ser Ala
Phe Gly Ser Met Lys Val Ser Arg Asp Lys 20 25 30 Asp Gly Ser Lys
Val Thr Thr Val Val Ala Thr Pro Gly Gln Gly Pro 35 40 45 Asp Arg
Pro Gln Glu Val Ser Tyr Thr Asp Thr Lys Val Ile Gly Asn 50 55 60
Gly Ser Phe Gly Val Val Tyr Gln Ala Lys Leu Cys Asp Ser Gly Glu 65
70 75 80 Leu Val Ala Ile Lys Lys Val Leu Gln Ala Lys Arg Phe Lys
Asn Arg 85 90 95 Glu Leu Gln Ile Met Arg Lys Leu Asp His Cys Asn
Ile Val Arg Leu 100 105 110 Arg Tyr Phe Phe Tyr Ser Ser Gly Glu Lys
Lys Asp Glu Val Tyr Leu 115 120 125 Asn Leu Val Leu Asp Tyr Val Pro
Glu Thr Val Tyr Arg Val Ala Arg 130 135 140 His Tyr Ser Arg Ala Lys
Gln Thr Leu Pro Val Ile Tyr Val Lys Leu 145 150 155 160 Tyr Met Tyr
Gln Leu Phe Arg Ser Leu Ala Tyr Ile His Ser Phe Gly 165 170 175 Ile
Cys His Arg Asp Ile Lys Pro Gln Asn Leu Leu Leu Asp Pro Asp 180 185
190 Thr Ala Val Leu Lys Leu Cys Asp Phe Gly Ser Ala Lys Gln Leu Val
195 200 205 Arg Gly Glu Pro Asn Val Ser Tyr Ile Cys Ser Arg Tyr Tyr
Arg Ala 210 215 220 Pro Glu Leu Ile Phe Gly Ala Thr Asp Tyr Thr Ser
Ser Ile Asp Val 225 230 235 240 Trp Ser Ala Gly Cys Val Leu Ala Glu
Leu Leu Leu Gly Gln Pro Ile 245 250 255 Phe Pro Gly Asp Ser Gly Val
Asp Gln Leu Val Glu Ile Ile Lys Val 260 265 270 Leu Gly Thr Pro Thr
Arg Glu Gln Ile Arg Glu Met Asn Pro Asn Tyr 275 280 285 Thr Glu Phe
Lys Phe Pro Gln Ile Lys Ala His Pro Trp Thr Lys Asp 290 295 300 Ser
Ser Gly Thr Gly His Phe Thr Ser Gly Val Arg Val Phe Arg Pro 305 310
315 320 Arg Thr Pro Pro Glu Ala Ile Ala Leu Cys Ser Arg Leu Leu Glu
Tyr 325 330 335 Thr Pro Thr Ala Arg Leu Thr Pro Leu Glu Ala Cys Ala
His Ser Phe 340 345 350 Phe Asp Glu Leu Arg Asp Pro Asn Val Lys Leu
Pro Asn Gly Arg Asp 355 360 365 Thr Pro Ala Leu Phe Asn Phe Thr Thr
Gln Glu Leu Ser Ser Asn Pro 370 375 380 Pro Leu Ala Thr Ile Leu Ile
Pro Pro His Ala Arg Ile Gln Ala Ala 385 390 395 400 Ala Ser Thr Pro
Thr Asn Ala Thr Ala Ala Ser Asp Ala Asn Thr Gly 405 410 415 Asp Arg
Gly Gln Thr Asn Asn Ala Ala Ser Ala Ser Ala Ser Asn Ser 420 425 430
Thr 7433PRTArtificial sequenceGSK-3 beta, K91A mutant 7Met Ser Gly
Arg Pro Arg Thr Thr Ser Phe Ala Glu Ser Cys Lys Pro 1 5 10 15 Val
Gln Gln Pro Ser Ala Phe Gly Ser Met Lys Val Ser Arg Asp Lys 20 25
30 Asp Gly Ser Lys Val Thr Thr Val Val Ala Thr Pro Gly Gln Gly Pro
35 40 45 Asp Arg Pro Gln Glu Val Ser Tyr Thr Asp Thr Lys Val Ile
Gly Asn 50 55 60 Gly Ser Phe Gly Val Val Tyr Gln Ala Lys Leu Cys
Asp Ser Gly Glu 65 70 75 80 Leu Val Ala Ile Lys Lys Val Leu Gln Asp
Ala Arg Phe Lys Asn Arg 85 90 95 Glu Leu Gln Ile Met Arg Lys Leu
Asp His Cys Asn Ile Val Arg Leu 100 105 110 Arg Tyr Phe Phe Tyr Ser
Ser Gly Glu Lys Lys Asp Glu Val Tyr Leu 115 120 125 Asn Leu Val Leu
Asp Tyr Val Pro Glu Thr Val Tyr Arg Val Ala Arg 130 135 140 His Tyr
Ser Arg Ala Lys Gln Thr Leu Pro Val Ile Tyr Val Lys Leu 145 150 155
160 Tyr Met Tyr Gln Leu Phe Arg Ser Leu Ala Tyr Ile His Ser Phe Gly
165 170 175 Ile Cys His Arg Asp Ile Lys Pro Gln Asn Leu Leu Leu Asp
Pro Asp 180 185 190 Thr Ala Val Leu Lys Leu Cys Asp Phe Gly Ser Ala
Lys Gln Leu Val 195 200 205 Arg Gly Glu Pro Asn Val Ser Tyr Ile Cys
Ser Arg Tyr Tyr Arg Ala 210 215 220 Pro Glu Leu Ile Phe Gly Ala Thr
Asp Tyr Thr Ser Ser Ile Asp Val 225 230 235 240 Trp Ser Ala Gly Cys
Val Leu Ala Glu Leu Leu Leu Gly Gln Pro Ile 245 250 255 Phe Pro Gly
Asp Ser Gly Val Asp Gln Leu Val Glu Ile Ile Lys Val 260 265 270 Leu
Gly Thr Pro Thr Arg Glu Gln Ile Arg Glu Met Asn Pro Asn Tyr 275 280
285 Thr Glu Phe Lys Phe Pro Gln Ile Lys Ala His Pro Trp Thr Lys Asp
290 295 300 Ser Ser Gly Thr Gly His Phe Thr Ser Gly Val Arg Val Phe
Arg Pro 305 310 315 320 Arg Thr Pro Pro Glu Ala Ile Ala Leu Cys Ser
Arg Leu Leu Glu Tyr 325 330 335 Thr Pro Thr Ala Arg Leu Thr Pro Leu
Glu Ala Cys Ala His Ser Phe 340 345 350 Phe Asp Glu Leu Arg Asp Pro
Asn Val Lys Leu Pro Asn Gly Arg Asp 355 360 365 Thr Pro Ala Leu Phe
Asn Phe Thr Thr Gln Glu Leu Ser Ser Asn Pro 370 375 380 Pro Leu Ala
Thr Ile Leu Ile Pro Pro His Ala Arg Ile Gln Ala Ala 385 390 395 400
Ala Ser Thr Pro Thr Asn Ala Thr Ala Ala Ser Asp Ala Asn Thr Gly 405
410 415 Asp Arg Gly Gln Thr Asn Asn Ala Ala Ser Ala Ser Ala Ser Asn
Ser 420 425 430 Thr 8433PRTArtificial sequenceGSK-3 beta, R92A
mutant 8Met Ser Gly Arg Pro Arg Thr Thr Ser Phe Ala Glu Ser Cys Lys
Pro 1 5 10 15 Val Gln Gln Pro Ser Ala Phe Gly Ser Met Lys Val Ser
Arg Asp Lys 20 25 30 Asp Gly Ser Lys Val Thr Thr Val Val Ala Thr
Pro Gly Gln Gly Pro 35 40 45 Asp Arg Pro Gln Glu Val Ser Tyr Thr
Asp Thr Lys Val Ile Gly Asn 50 55 60 Gly Ser Phe Gly Val Val Tyr
Gln Ala Lys Leu Cys Asp Ser Gly Glu 65 70 75 80 Leu Val Ala Ile Lys
Lys Val Leu Gln Asp Lys Ala Phe Lys Asn Arg 85 90 95 Glu Leu Gln
Ile Met Arg Lys Leu Asp His Cys Asn Ile Val Arg Leu 100 105 110 Arg
Tyr Phe Phe Tyr Ser Ser Gly Glu Lys Lys Asp Glu Val Tyr Leu 115 120
125 Asn Leu Val Leu Asp Tyr Val Pro Glu Thr Val Tyr Arg Val Ala Arg
130 135 140 His Tyr Ser Arg Ala Lys Gln Thr Leu Pro Val Ile Tyr Val
Lys Leu 145 150 155 160 Tyr Met Tyr Gln Leu Phe Arg Ser Leu Ala Tyr
Ile His Ser Phe Gly 165 170 175 Ile Cys His Arg Asp Ile Lys Pro Gln
Asn Leu Leu Leu Asp Pro Asp 180 185 190 Thr Ala Val Leu Lys Leu Cys
Asp Phe Gly Ser Ala Lys Gln Leu Val 195 200 205 Arg Gly Glu Pro Asn
Val Ser Tyr Ile Cys Ser Arg Tyr Tyr Arg Ala 210 215 220 Pro Glu Leu
Ile Phe Gly Ala Thr Asp Tyr Thr Ser Ser Ile Asp Val 225 230 235 240
Trp Ser Ala Gly Cys Val Leu Ala Glu Leu Leu Leu Gly Gln Pro Ile 245
250 255 Phe Pro Gly Asp Ser Gly Val Asp Gln Leu Val Glu Ile Ile Lys
Val 260 265 270 Leu Gly Thr Pro Thr Arg Glu Gln Ile Arg Glu Met Asn
Pro Asn Tyr 275 280 285 Thr Glu Phe Lys Phe Pro Gln Ile Lys Ala His
Pro Trp Thr Lys Asp 290 295 300 Ser Ser Gly Thr Gly His Phe Thr Ser
Gly Val Arg Val Phe Arg Pro 305 310 315 320 Arg Thr Pro Pro Glu Ala
Ile Ala Leu Cys Ser Arg Leu Leu Glu Tyr 325 330 335 Thr Pro Thr Ala
Arg Leu Thr Pro Leu Glu Ala Cys Ala His Ser Phe 340 345 350 Phe Asp
Glu Leu Arg Asp Pro Asn Val Lys Leu Pro Asn Gly Arg Asp 355 360 365
Thr Pro Ala Leu Phe Asn Phe Thr Thr Gln Glu Leu Ser Ser Asn Pro 370
375 380 Pro Leu Ala Thr Ile Leu Ile Pro Pro His Ala Arg Ile Gln Ala
Ala 385 390 395 400 Ala Ser Thr Pro Thr Asn Ala Thr Ala Ala Ser Asp
Ala Asn Thr Gly 405 410 415 Asp Arg Gly Gln Thr Asn Asn Ala Ala Ser
Ala Ser Ala Ser Asn Ser 420 425 430 Thr 9433PRTArtificial
sequenceGSK-3 beta, F93A mutant 9Met Ser Gly Arg Pro Arg Thr Thr
Ser Phe Ala Glu Ser Cys Lys Pro 1 5 10 15 Val Gln Gln Pro Ser Ala
Phe Gly Ser Met Lys Val Ser Arg Asp Lys 20 25 30 Asp Gly Ser Lys
Val Thr Thr Val Val Ala Thr Pro Gly Gln Gly Pro 35 40 45 Asp Arg
Pro Gln Glu Val Ser Tyr Thr Asp Thr Lys Val Ile Gly Asn 50 55 60
Gly Ser Phe Gly Val Val Tyr Gln Ala Lys Leu Cys Asp Ser Gly Glu 65
70 75 80 Leu Val Ala Ile Lys Lys Val Leu Gln Asp Lys Arg Ala Lys
Asn Arg 85 90 95 Glu Leu Gln Ile Met Arg Lys Leu Asp His Cys Asn
Ile Val Arg Leu 100 105 110 Arg Tyr Phe Phe Tyr Ser Ser Gly Glu Lys
Lys Asp Glu Val Tyr Leu 115 120 125 Asn Leu Val Leu Asp Tyr Val Pro
Glu Thr Val Tyr Arg Val Ala Arg 130 135 140 His Tyr Ser Arg Ala Lys
Gln Thr Leu Pro Val Ile Tyr Val Lys Leu 145 150 155 160 Tyr Met Tyr
Gln Leu Phe Arg Ser Leu Ala Tyr Ile His Ser Phe Gly 165 170 175 Ile
Cys His Arg Asp Ile Lys Pro Gln Asn Leu Leu Leu Asp Pro Asp 180 185
190 Thr Ala Val Leu Lys Leu Cys Asp Phe Gly Ser Ala Lys Gln Leu Val
195 200 205 Arg Gly Glu Pro Asn Val Ser Tyr Ile Cys Ser Arg Tyr Tyr
Arg Ala 210 215 220 Pro Glu Leu Ile Phe Gly Ala Thr Asp Tyr Thr Ser
Ser Ile Asp Val 225 230 235 240 Trp Ser Ala Gly Cys Val Leu Ala Glu
Leu Leu Leu Gly Gln Pro Ile 245 250 255 Phe Pro Gly Asp Ser Gly Val
Asp Gln Leu Val Glu Ile Ile Lys Val 260 265 270 Leu Gly Thr Pro Thr
Arg Glu Gln Ile Arg Glu Met Asn Pro Asn Tyr 275 280 285 Thr Glu Phe
Lys Phe Pro Gln Ile Lys Ala His Pro Trp Thr Lys Asp 290 295 300 Ser
Ser Gly Thr Gly His Phe Thr Ser Gly Val Arg Val Phe Arg Pro 305 310
315 320 Arg Thr Pro Pro Glu Ala Ile Ala Leu Cys Ser Arg Leu Leu Glu
Tyr 325 330 335 Thr Pro Thr Ala Arg Leu Thr Pro Leu Glu Ala Cys Ala
His Ser Phe 340 345 350 Phe Asp Glu Leu Arg Asp Pro Asn Val Lys Leu
Pro Asn Gly Arg Asp 355 360 365 Thr Pro Ala Leu Phe Asn Phe Thr Thr
Gln Glu Leu Ser Ser Asn Pro 370 375 380 Pro Leu Ala Thr Ile Leu Ile
Pro Pro His Ala Arg Ile Gln Ala Ala 385 390 395 400 Ala Ser Thr Pro
Thr Asn Ala Thr Ala Ala Ser Asp Ala Asn Thr Gly 405 410 415 Asp Arg
Gly Gln Thr Asn Asn Ala Ala Ser Ala Ser Ala Ser Asn Ser 420 425 430
Thr 10433PRTArtificial sequenceGSK-3 beta, K94A mutant 10Met Ser
Gly Arg Pro Arg Thr Thr Ser Phe Ala Glu Ser Cys Lys Pro 1 5 10 15
Val Gln Gln Pro Ser Ala Phe Gly Ser Met Lys Val Ser Arg Asp Lys 20
25 30 Asp Gly Ser Lys Val Thr Thr Val Val
Ala Thr Pro Gly Gln Gly Pro 35 40 45 Asp Arg Pro Gln Glu Val Ser
Tyr Thr Asp Thr Lys Val Ile Gly Asn 50 55 60 Gly Ser Phe Gly Val
Val Tyr Gln Ala Lys Leu Cys Asp Ser Gly Glu 65 70 75 80 Leu Val Ala
Ile Lys Lys Val Leu Gln Asp Lys Arg Phe Ala Asn Arg 85 90 95 Glu
Leu Gln Ile Met Arg Lys Leu Asp His Cys Asn Ile Val Arg Leu 100 105
110 Arg Tyr Phe Phe Tyr Ser Ser Gly Glu Lys Lys Asp Glu Val Tyr Leu
115 120 125 Asn Leu Val Leu Asp Tyr Val Pro Glu Thr Val Tyr Arg Val
Ala Arg 130 135 140 His Tyr Ser Arg Ala Lys Gln Thr Leu Pro Val Ile
Tyr Val Lys Leu 145 150 155 160 Tyr Met Tyr Gln Leu Phe Arg Ser Leu
Ala Tyr Ile His Ser Phe Gly 165 170 175 Ile Cys His Arg Asp Ile Lys
Pro Gln Asn Leu Leu Leu Asp Pro Asp 180 185 190 Thr Ala Val Leu Lys
Leu Cys Asp Phe Gly Ser Ala Lys Gln Leu Val 195 200 205 Arg Gly Glu
Pro Asn Val Ser Tyr Ile Cys Ser Arg Tyr Tyr Arg Ala 210 215 220 Pro
Glu Leu Ile Phe Gly Ala Thr Asp Tyr Thr Ser Ser Ile Asp Val 225 230
235 240 Trp Ser Ala Gly Cys Val Leu Ala Glu Leu Leu Leu Gly Gln Pro
Ile 245 250 255 Phe Pro Gly Asp Ser Gly Val Asp Gln Leu Val Glu Ile
Ile Lys Val 260 265 270 Leu Gly Thr Pro Thr Arg Glu Gln Ile Arg Glu
Met Asn Pro Asn Tyr 275 280 285 Thr Glu Phe Lys Phe Pro Gln Ile Lys
Ala His Pro Trp Thr Lys Asp 290 295 300 Ser Ser Gly Thr Gly His Phe
Thr Ser Gly Val Arg Val Phe Arg Pro 305 310 315 320 Arg Thr Pro Pro
Glu Ala Ile Ala Leu Cys Ser Arg Leu Leu Glu Tyr 325 330 335 Thr Pro
Thr Ala Arg Leu Thr Pro Leu Glu Ala Cys Ala His Ser Phe 340 345 350
Phe Asp Glu Leu Arg Asp Pro Asn Val Lys Leu Pro Asn Gly Arg Asp 355
360 365 Thr Pro Ala Leu Phe Asn Phe Thr Thr Gln Glu Leu Ser Ser Asn
Pro 370 375 380 Pro Leu Ala Thr Ile Leu Ile Pro Pro His Ala Arg Ile
Gln Ala Ala 385 390 395 400 Ala Ser Thr Pro Thr Asn Ala Thr Ala Ala
Ser Asp Ala Asn Thr Gly 405 410 415 Asp Arg Gly Gln Thr Asn Asn Ala
Ala Ser Ala Ser Ala Ser Asn Ser 420 425 430 Thr 1111PRTArtificial
sequencePK1A synthetic peptide 11Ala Glu Ala Pro Pro Ala Pro Pro
Gln Ser Pro 1 5 10 1211PRTArtificial sequencePE2A synthetic peptide
12Lys Ala Ala Pro Pro Ala Pro Pro Gln Ser Pro 1 5 10
1311PRTArtificial sequencePQ9A synthetic peptide 13Lys Glu Ala Pro
Pro Ala Pro Pro Ala Ser Pro 1 5 10 1411PRTArtificial sequencePQ9R
synthetic peptide 14Lys Glu Ala Pro Pro Ala Pro Pro Arg Ser Pro 1 5
10 1511PRTArtificial sequencePQ9Y synthetic peptide 15Lys Glu Ala
Pro Pro Ala Pro Pro Tyr Ser Pro 1 5 10 1611PRTArtificial
sequencePQ9P (L806) synthetic peptide 16Lys Glu Ala Pro Pro Ala Pro
Pro Pro Ser Pro 1 5 10 1712PRTArtificial sequencePQ9P+myristic acid
at the N-terminus (L806-mts) synthetic peptide 17Gly Lys Glu Ala
Pro Pro Ala Pro Pro Pro Ser Pro 1 5 10 189PRTArtificial
sequencep9CREB synthetic peptide 18Ile Leu Ser Arg Arg Pro Ser Tyr
Arg 1 5 1914PRTArtificial sequencepIRS-1 synthetic peptide 19Arg
Arg Glu Gly Gly Met Ser Arg Pro Ala Ser Val Asp Gly 1 5 10
2026PRTArtificial sequencePGS-1 synthetic peptide 20Tyr Arg Arg Ala
Ala Val Pro Pro Ser Pro Ser Leu Ser Arg His Ser 1 5 10 15 Ser Pro
Ser Gln Ser Glu Asp Glu Glu Glu 20 25 215PRTArtificial sequenceAn
exemplary portion of the peptide describe in the prefered
embodimets 21Ala Pro Pro Pro Ser 1 5 225PRTArtificial sequenceAn
exemplary portion of the peptide describe in the prefered
embodimets 22Ala Pro Pro Pro Thr 1 5 235PRTArtificial sequenceAn
exemplary portion of the peptide describe in the prefered
embodimets 23Ala Ala Pro Pro Ser 1 5 245PRTArtificial sequenceAn
exemplary portion of the peptide describe in the prefered
embodimets 24Ala Ala Pro Pro Thr 1 5 255PRTArtificial sequenceAn
exemplary portion of the peptide describe in the prefered
embodimets 25Ala Ala Ala Pro Ser 1 5 265PRTArtificial sequenceAn
exemplary portion of the peptide describe in the prefered
embodimets 26Ala Ala Ala Pro Thr 1 5 275PRTArtificial sequenceAn
exemplary portion of the peptide describe in the prefered
embodimets 27Ala Pro Ala Pro Ser 1 5 285PRTArtificial sequenceAn
exemplary portion of the peptide describe in the prefered
embodimets 28Ala Pro Ala Pro Thr 1 5 295PRTArtificial sequenceAn
exemplary portion of the peptide describe in the prefered
embodimets 29Ala Gly Pro Pro Ser 1 5 305PRTArtificial sequenceAn
exemplary portion of the peptide describe in the prefered
embodimets 30Ala Gly Pro Pro Thr 1 5 315PRTArtificial sequenceAn
exemplary portion of the peptide describe in the prefered
embodimets 31Ala Gly Gly Pro Ser 1 5 325PRTArtificial sequenceAn
exemplary portion of the peptide describe in the prefered
embodimets 32Ala Gly Gly Pro Thr 1 5 335PRTArtificial sequenceAn
exemplary portion of the peptide describe in the prefered
embodimets 33Ala Pro Gly Pro Ser 1 5 345PRTArtificial sequenceAn
exemplary portion of the peptide describe in the prefered
embodimets 34Ala Pro Gly Pro Thr 1 5 355PRTArtificial sequenceAn
exemplary portion of the peptide describe in the prefered
embodimets 35Ala Xaa Pro Pro Ser 1 5 365PRTArtificial sequenceAn
exemplary portion of the peptide describe in the prefered
embodimets 36Ala Xaa Pro Pro Thr 1 5 375PRTArtificial sequenceAn
exemplary portion of the peptide describe in the prefered
embodimets 37Ala Xaa Xaa Pro Ser 1 5 385PRTArtificial sequenceAn
exemplary portion of the peptide describe in the prefered
embodimets 38Ala Xaa Xaa Pro Thr 1 5 395PRTArtificial sequenceAn
exemplary portion of the peptide describe in the prefered
embodimets 39Ala Pro Xaa Pro Ser 1 5 405PRTArtificial sequenceAn
exemplary portion of the peptide describe in the prefered
embodimets 40Ala Pro Xaa Pro Thr 1 5 415PRTArtificial sequenceAn
exemplary portion of the peptide describe in the prefered
embodimets 41Ala Val Pro Pro Ser 1 5 425PRTArtificial sequenceAn
exemplary portion of the peptide describe in the prefered
embodimets 42Ala Val Pro Pro Thr 1 5 435PRTArtificial sequenceAn
exemplary portion of the peptide describe in the prefered
embodimets 43Ala Val Val Pro Ser 1 5 445PRTArtificial sequenceAn
exemplary portion of the peptide describe in the prefered
embodimets 44Ala Val Val Pro Thr 1 5 455PRTArtificial sequenceAn
exemplary portion of the peptide describe in the prefered
embodimets 45Ala Pro Val Pro Ser 1 5 465PRTArtificial sequenceAn
exemplary portion of the peptide describe in the prefered
embodimets 46Ala Pro Val Pro Thr 1 5 47433PRTArtificial
sequenceGSK-3 beta, V214A mutant 47Met Ser Gly Arg Pro Arg Thr Thr
Ser Phe Ala Glu Ser Cys Lys Pro 1 5 10 15 Val Gln Gln Pro Ser Ala
Phe Gly Ser Met Lys Val Ser Arg Asp Lys 20 25 30 Asp Gly Ser Lys
Val Thr Thr Val Val Ala Thr Pro Gly Gln Gly Pro 35 40 45 Asp Arg
Pro Gln Glu Val Ser Tyr Thr Asp Thr Lys Val Ile Gly Asn 50 55 60
Gly Ser Phe Gly Val Val Tyr Gln Ala Lys Leu Cys Asp Ser Gly Glu 65
70 75 80 Leu Val Ala Ile Lys Lys Val Leu Gln Asp Lys Arg Phe Lys
Asn Arg 85 90 95 Glu Leu Gln Ile Met Arg Lys Leu Asp His Cys Asn
Ile Val Arg Leu 100 105 110 Arg Tyr Phe Phe Tyr Ser Ser Gly Glu Lys
Lys Asp Glu Val Tyr Leu 115 120 125 Asn Leu Val Leu Asp Tyr Val Pro
Glu Thr Val Tyr Arg Val Ala Arg 130 135 140 His Tyr Ser Arg Ala Lys
Gln Thr Leu Pro Val Ile Tyr Val Lys Leu 145 150 155 160 Tyr Met Tyr
Gln Leu Phe Arg Ser Leu Ala Tyr Ile His Ser Phe Gly 165 170 175 Ile
Cys His Arg Asp Ile Lys Pro Gln Asn Leu Leu Leu Asp Pro Asp 180 185
190 Thr Ala Val Leu Lys Leu Cys Asp Phe Gly Ser Ala Lys Gln Leu Val
195 200 205 Arg Gly Glu Pro Asn Ala Ser Tyr Ile Cys Ser Arg Tyr Tyr
Arg Ala 210 215 220 Pro Glu Leu Ile Phe Gly Ala Thr Asp Tyr Thr Ser
Ser Ile Asp Val 225 230 235 240 Trp Ser Ala Gly Cys Val Leu Ala Glu
Leu Leu Leu Gly Gln Pro Ile 245 250 255 Phe Pro Gly Asp Ser Gly Val
Asp Gln Leu Val Glu Ile Ile Lys Val 260 265 270 Leu Gly Thr Pro Thr
Arg Glu Gln Ile Arg Glu Met Asn Pro Asn Tyr 275 280 285 Thr Glu Phe
Lys Phe Pro Gln Ile Lys Ala His Pro Trp Thr Lys Asp 290 295 300 Ser
Ser Gly Thr Gly His Phe Thr Ser Gly Val Arg Val Phe Arg Pro 305 310
315 320 Arg Thr Pro Pro Glu Ala Ile Ala Leu Cys Ser Arg Leu Leu Glu
Tyr 325 330 335 Thr Pro Thr Ala Arg Leu Thr Pro Leu Glu Ala Cys Ala
His Ser Phe 340 345 350 Phe Asp Glu Leu Arg Asp Pro Asn Val Lys Leu
Pro Asn Gly Arg Asp 355 360 365 Thr Pro Ala Leu Phe Asn Phe Thr Thr
Gln Glu Leu Ser Ser Asn Pro 370 375 380 Pro Leu Ala Thr Ile Leu Ile
Pro Pro His Ala Arg Ile Gln Ala Ala 385 390 395 400 Ala Ser Thr Pro
Thr Asn Ala Thr Ala Ala Ser Asp Ala Asn Thr Gly 405 410 415 Asp Arg
Gly Gln Thr Asn Asn Ala Ala Ser Ala Ser Ala Ser Asn Ser 420 425 430
Thr
* * * * *