U.S. patent application number 13/251390 was filed with the patent office on 2013-02-14 for methods and means for increasing resistance to cell damage.
This patent application is currently assigned to UNIVERSITY OF MEDICINE AND DENTISTRY OF NEW JERSEY. The applicant listed for this patent is Hsueh-Ping Chu, Alexey G. Ryazanov. Invention is credited to Hsueh-Ping Chu, Alexey G. Ryazanov.
Application Number | 20130041009 13/251390 |
Document ID | / |
Family ID | 35782381 |
Filed Date | 2013-02-14 |
United States Patent
Application |
20130041009 |
Kind Code |
A1 |
Ryazanov; Alexey G. ; et
al. |
February 14, 2013 |
Methods and means for increasing resistance to cell damage
Abstract
Methods are provided to increase resistance to cell damage in a
subject. The increase in resistance to cell damage in a subject in
the subject is accomplished by decreasing activity of eEF2 kinase
in the subject. The eEF2 kinase activity can be decreased by
decreasing the amount of functional eEF2 kinase produced by the
subject, including contacting the eEF2 kinase with a compound that
inhibits phosphorylation of eEF2 kinase substrate or decreasing the
amount of functional eEF2 kinase is decreased by reducing
expression of a gene encoding the eEF2 kinase.
Inventors: |
Ryazanov; Alexey G.;
(Princeton, NJ) ; Chu; Hsueh-Ping; (Taipei,
TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ryazanov; Alexey G.
Chu; Hsueh-Ping |
Princeton
Taipei |
NJ |
US
TW |
|
|
Assignee: |
UNIVERSITY OF MEDICINE AND
DENTISTRY OF NEW JERSEY
Somerset
NJ
|
Family ID: |
35782381 |
Appl. No.: |
13/251390 |
Filed: |
October 3, 2011 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11615690 |
Dec 22, 2006 |
8030286 |
|
|
13251390 |
|
|
|
|
PCT/US2005/022741 |
Jun 24, 2005 |
|
|
|
11615690 |
|
|
|
|
60582411 |
Jun 24, 2004 |
|
|
|
60819688 |
Jul 10, 2006 |
|
|
|
Current U.S.
Class: |
514/44A ;
514/44R |
Current CPC
Class: |
A61P 43/00 20180101;
A01K 67/0276 20130101; A01K 2267/03 20130101; A01K 2217/075
20130101; C12N 15/8509 20130101; A01K 2227/105 20130101; A61K
31/675 20130101; C12N 2799/027 20130101; C12N 9/1205 20130101 |
Class at
Publication: |
514/44.A ;
514/44.R |
International
Class: |
A61K 31/713 20060101
A61K031/713; A61P 43/00 20060101 A61P043/00; A61K 31/7105 20060101
A61K031/7105 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] The U.S. Government has a paid-up license in this invention
and the right in limited circumstances to require the patent owner
to license others on reasonable terms as provided for by the terms
of grant R01AG19890 awarded by the U.S. National Institutes of
Health (National Institute on Aging).
Claims
1. A method of increasing resistance to cell damage in a subject
caused by a cytotoxic agent, the method comprising identifying a
subject that has been, or is suspected of having been, or is
expected to be, exposed to a cytotoxic agent, administering to the
subject an amount of a compound that decreases phosphorylation of
an eEF2 kinase substrate by an eEF2 kinase, wherein said amount is
effective to increase protein turnover in a cell of the subject at
a rate effective to increase resistance to cell damage.
2. The method of claim 1, wherein said phosphorylation of said eEF2
kinase substrate is decreased by decreasing eEF2 kinase catalytic
activity in said cell.
3. The method of claim 2, wherein said eEF2 kinase catalytic
activity is decreased by contacting the eEF2 kinase with the
compound.
4. The method of claim 4, wherein said phosphorylation of the eEF2
kinase substrate is decreased by reducing expression of a gene
encoding the eEF2 kinase in said cell.
5. The method of claim 4, wherein said expression of the gene
encoding the eEF2 kinase is reduced by altering said gene such that
the gene encodes a dysfunctional or non-functional eEF2 kinase.
6. The method of claim 4, wherein said expression of the gene
encoding the eEF2 kinase is reduced by contacting said gene, or an
mRNA transcribed from said gene, with the compound, the compound
comprising a polynucleotide selected from the group consisting of
an antisense oligonucleotide, a ribozyme, an siRNA, and an
shRNA.
7. The method of claim 6, wherein said compound comprises a
polynucleotide comprising a nucleotide sequence complementary to a
nucleotide sequence encoding a polypeptide comprising the amino
acid sequence of SEQ ID NO: 2.
8. The method of claim 7, wherein said compound comprises a
nucleotide sequence complementary to a nucleotide sequence
comprising the nucleotide sequence of SEQ ID NO: 1.
9. The method of claim 4, wherein the cytotoxic agent is selected
from the group consisting of treatment increased acidity, oxidative
stress, a chemotherapy agent, ionizing radiation, ultraviolet
radiation, and free radicals.
10. The method of claim 9, wherein the chemotherapy agent is
camptothecin (CPT), doxorubicin (DOX), or taxol.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. patent
application Ser. No. 11/615,690, filed Dec. 22, 2006, which is a
continuation-in-part of International Patent Application Serial No.
PCT/US2005/022741, filed Jun. 24, 2005, which claims priority to
U.S. Provisional Patent Application Ser. No. 60/582,411, filed on
Jun. 24, 2004. U.S. patent application Ser. No. 11/615,690 also
claims priority under 35 U.S.C. .sctn.119(e) to U.S. Provisional
Application Ser. No. 60/819,688, which was filed on Jul. 10, 2006.
The disclosures of all of the foregoing applications are hereby
incorporated by reference in their entireties.
FIELD OF THE INVENTION
[0003] The present invention relates to the field of cell damage
and the development of compositions and methods to increase
resistance to cell damage. In particular, the invention relates to
the manipulation of the elongation factor 2 (eEF2) kinase in order
to increase resistance to cell damage and increase life span of
cells.
BACKGROUND OF THE INVENTION
[0004] Accumulation of damaged cellular proteins is postulated to
be a major contributor to aging and sensitivity to cell damage.
Decreases in both protein synthesis and degradation rates may
result in the persistence of defective or modified proteins and
thus the overall rate of protein turnover can affect the cells'
response to cell damage.
[0005] Elongation factor-2 kinase (eEF2 kinase) is a ubiquitous
protein kinase that belongs to the alpha-kinase family of protein
kinases. these protein kinases do not display sequence homology to
conventional eukaryotic protein kinases (Ryazanov, A. G. et al.
Proc. Natl. Acad. Sci. USA 94, 4884-4889 (1997); Ryazanov, A. G.,
Pavur, K. S., Dorovkov, M. V. Curr. Biol. 28, 943-945 (1999); and
Ryazanov, A. G. FEBS Lett. 514, 26-29 (2002)). The only known
substrate of eEF2 kinase is eEF2, the protein that promotes
ribosomal translocation during the elongation phase of protein
synthesis. eEF2 is inactivated upon phosphorylation by eEF2 kinase,
implying that the phosphorylation process catalyzed by eEF2 kinase
can be a mechanism of protein synthesis regulation at the
elongation stage (Ryazanov, A. G., et al. Nature 334, 170-173
(1988)). eEF2 kinase is a highly specific protein kinase, which
phosphorylates and inactivates elongation factor-2 (Ryazanov, A.
G., et al. Nature 324, 170-173 (1988); and Ryazanov, A. G. et al.
Proc. Natl. Acad. Sci. U.S.A. 94, 4884-4889 (1997)). This kinase is
regulated by the IGF-1/mTOR pathway and is implicated in the
control of the global rate of protein synthesis at the elongation
stage (Browne, G. J., Proud C. G. Eur. J. Biochem. 269, 5360-5368
(2002); and Proud C. G. Curr. Top. Microbiol. Immunol. 279, 215-244
(2004)). eEF2 kinase activity in cells is normally low, but is
stimulated by various stress-inducing agents such as Ca.sup.2+
ionophores, acidic pH and hydrogen peroxide (Nairn, A. C. and
Palfrey, H. C. Cold Spring Harbor Laboratory Press, 295-318 (1996);
Patel, J. et al., Eur. J. Biochem. 269, 3076-3085 (2002); and
Dorovkov, M. V., et al. Biochemistry 41, 13444-13450 (2002)).
[0006] To uncover the physiological role of eEF2K eEF2K knockout
mice are prepared. Despite a complete lack of eEF2K activity, eEF2K
knockout mice have normal development, behavior and reproduction.
Moreover, these mice have increased lifespan. However, fibroblasts
from eEF2K knockout mice are found to be resistant to various
cytotoxic agents. The effect of eEF2K knockout on cell resistance
to cytotoxic agents may depend on functional p53 since it is
abolished in cells in which p53 is inactivated. Intriguingly,
knockout mice have significantly extended maximal lifespan. These
findings suggest that eEF2K is a modulator of stress resistance and
aging, and that its inactivation could protect cells from
stress-induced injury and increase life span in mammals.
[0007] One of the hallmarks of aging is the progressive decline in
the rate of protein synthesis and degradation. This decline in
protein turnover can be a major factor contributing to an increase
in the concentration of damaged proteins with age. Therefore, by
regulating the overall rate of protein synthesis and/or degradation
it might be possible to modulate the rate of aging. Since knockout
of eEF2 kinase increases maximal life span in mice, manipulation of
expression or activity if eEF2 kinase may offer a therapeutic basis
for regulating protein turnover and reducing cell damage due to
stress, exposure to chemotherapeutic agents, and other factors.
SUMMARY OF THE INVENTION
[0008] In accordance with the present invention, it has now been
shown for the first time that a decrease in activity of eEF2 kinase
causes an increase in overall protein turnover in cells, the result
being increased resistance to cell damage, particularly
stress-induced cell damage. Since the same enzyme is present in all
animals, including humans, it is now clearly predictable that
increasing protein turnover in cells, particularly through the
inhibition of this enzyme, in human and animal subjects will result
in decreasing cell death. The discoveries made in accordance with
the present invention enable a variety of useful methods, kits and
pharmaceutical formulations directed to increasing resistance to
cell damage in the cells of a subject, thereby decreasing cell
death.
[0009] According to one aspect of the invention, methods are
provided to increase resistance to cell damage in a subject. These
methods comprise increasing protein turnover in the subject, the
increase in protein turnover resulting in the increased resistance
to cell damage of the subject. Preferably, the protein turnover in
the subject is accomplished by decreasing activity of eEF2 kinase
in the subject. In one embodiment, the eEF2 kinase activity is
decreased by decreasing the amount of functional eEF2 kinase
produced by the subject. In a preferred embodiment the amount of
functional eEF2 kinase is decreased by decreasing eEF2 kinase
activity in the cell, preferably by contacting the eEF2 kinase with
a compound that inhibits phosphorylation of eEF2. In another
preferred embodiment, the amount of functional eEF2 kinase is
decreased by reducing expression of a gene encoding the eEF2
kinase. Alternatively, the amount of functional eEF2 kinase is
decreased by altering a gene encoding the eEF2 kinase such that the
gene encodes a dysfunctional or non-functional eEF2 kinase.
[0010] The invention also provides a method of increasing
resistance to cell damage in a subject caused by a cytotoxic agent.
The method includes identifying a subject that has been, or is
suspected of having been, or is expected to be, exposed to a
cytotoxic agent; administering to the subject an amount of a
compound that decreases phosphorylation of an eEF2 kinase substrate
by an eEF2 kinase. The amount is effective to increase protein
turnover in a cell of the subject at a rate effective to increase
resistance to cell damage. In one embodiment, the phosphorylation
of the eEF2 kinase substrate is decreased by decreasing eEF2 kinase
catalytic activity in the cell. In another embodiment, the eEF2
kinase catalytic activity is decreased by contacting the eEF2
kinase with the compound. In yet another embodiment, the
phosphorylation of the eEF2 kinase substrate is decreased by
reducing expression of a gene encoding the eEF2 kinase in the cell.
The expression of the gene encoding the eEF2 kinase can be reduced
by altering the gene such that the gene encodes a dysfunctional or
non-functional eEF2 kinase. Alternatively, the expression of the
gene encoding the eEF2 kinase can be reduced by contacting the
gene, or an mRNA transcribed from the gene, with the compound that
contains a polynucleotide selected from the group consisting of an
antisense oligonucleotide, a ribozyme, an siRNA, and an shRNA. In
one example, the compound has a polynucleotide containing a
nucleotide sequence complementary to a nucleotide sequence encoding
a polypeptide comprising the amino acid sequence of SEQ ID NO: 2,
such as a nucleotide sequence complementary to a nucleotide
sequence having the nucleotide sequence of SEQ ID NO: 1. In the
above-disclosed method, the cytotoxic agent can be selected from
the group consisting of treatment increased acidity, oxidative
stress, a chemotherapy agent, ionizing radiation, ultraviolet
radiation, and free radicals. Examples of the chemotherapy agent
include camptothecin (CPT), doxorubicin (DOX), and taxol.
[0011] According to another aspect of the invention, a genetically
manipulated non-human organism is provided, in which an enzyme that
negatively regulates protein synthesis is dysfunctional,
non-functional or absent. Preferably, the organism is a rodent,
preferably a mouse and the enzyme is eEF2 kinase. Another aspect of
the present invention relates to vector constructs useful in the
construction of genetically manipulated non-human organism, in
which eEF2 kinase is dysfunctional, non-functional or absent.
[0012] According to another aspect of the invention, a
pharmaceutical formulation for increasing resistance to cell damage
of a subject is provided. The formulation comprises an agent that
increases cellular protein turnover in a biologically compatible
medium. Preferably, the formulation comprises an inhibitor of eEF2
activity or gene expression.
[0013] Various diagnostic and prognostic assays and kits are also
provided in accordance with the present invention, as described in
greater detail below. Other features and advantages of the present
invention will be understood by reference to the drawings, detailed
description and examples that follow.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1. Ionomycin, acidic pH and hydrogen peroxide
cytotoxicity is reduced in eEF2 kinase deficient cells. a. Map of
the wild type eEF2K locus, the targeting vector and the mutant
eEF2K locus after homologous recombination. b. Schematic
representation of wild type eEF2K and predicted truncated eEF2K
gene in eEF2K-/- mice. c. Western blot analysis of eEF2
phosphorylation in liver extracts from eEF2K+/+ and eEF2K-/- mice.
d. MTT viability assay for eEF2K+/+ (WT) and eEF2K-/- (KO) MEFs
after treatment with ionomycin at indicated concentrations for 24
hours. e. Graphs of MTT assay for eEF2K+/+ and eEF2K-/- MEFs
incubated at indicated pH for 3 hours. f. Graphs of MTT assay for
eEF2K+/+ and eEF2K-/- MEFs incubated with H2O2 at indicated
concentrations for 12 hours. g. Colony formation assay of eEF2K+/+
and eEF2K-/- stable cell lines after incubation at pH6 for 24
h.
[0015] FIG. 2. Generation of eEF2 kinase knockout in mice. (A) The
eEF2 gene knockout mice are generated by genomic targeting to the
germline transmission. In the eEF2 kinase gene knockout
construction, by the homologous recombination, the exon 7 and the
majority of exon 8 is replaced by Neo gene cassette that disrupted
the catalytic domain of eEF2 kinase. (B) This strategy resulted in
truncation of eEF2 kinase and elimination of subdomains IV-1.times.
from its catalytic domain (see Drennan and Ryazanov (2004) Progress
in Biophys & Mol. Biol. 85: 1-32 for designation of domains).
The correctly targeted ES cell lines are microinjected into
C57BL/6J and BaLA/cj host blastocysts. The chimeric mice are
generated and they give germline transmission of the disrupted eEF2
gene. (C) The genotypes of mice are inspected by PCR reactions
using primer pairs SA8/Neo1 to identify knockout allele and primer
pairs SA8/SA5 to detect WT allele (Neo1: 5'-TGC GAG GCC AGA GGC CAC
TTG TGT AGC-3' (SEQ ID NO: 5), SA8: 5'-GGC CGG CTG CTA GAG AGT
GTC-3' (SEQ ID NO: 6), SA5: 5'-CAT CAG CTG ATT GTA GTG GAC ATC-3'
(SEQ ID NO: 7)).
[0016] FIG. 3. eEF2 kinase deficiency increases the resistance of
mouse embryonic fibroblasts to chemotherapeutic drugs. a, b. Graphs
representing the response of eEF2+/+ (WT) and eEF2K-/- (KO) MEFs to
CPT and DOX, measured by MTT assay. Cells are incubated for 24 h at
indicated concentrations of a drug. c. Graphs of MTT assay
comparing the response of eEF2K+/+, eEF2K-/- and KO (eEF2K) (cell
line, transfected with cDNA of eEF2K) to doxorubicin. Experiment
performed as described in a, b. d. Flow cytometric analysis of
eEF2K+/+ and eEF2K-/- MEFs treated with 600 ng/ml of DOX for 24 h.
e. Analysis of apoptotic DNA fragmentation in wild type and
eEF2K-/- stable cell lines in response to a serum starvation (SS)
for 48 h.
[0017] FIG. 4. Effect of eEF2 kinase knockout on drug resistance
depends on functional p53. a. Drug sensitivity assay of eEF2K+/+
and eEF2K-/- stable cell lines and the same cell lines expressing
dominant-negative p53 mutant GSE56. Cells are treated with
indicated concentrations of DOX and after 24 h cell viability is
measured by MTT assay. b. Drug sensitivity assay of eEF2K+/+ and
eEF2K-/- stable cell lines and the same cell lines treated with PFT
a. MTT assay is done as described in a. c. Western blot analysis of
p53, p21 and 13 tubulin expression in wild type and eEF2K-/- MEFs
after DOX treatment. d. Quantification of the amount of p21 protein
before and after incubation with DOX.
[0018] FIG. 5. Knockout of eEF2 kinase leads to increased maximal
life span in mice. a. Comparison of survival curves of eEF2k-/-
(diamonds, n=82) and eEF2k+/+(circles, n=32) mice. b. Comparison of
survival curves of eEF2k+/-(triangles, n=38) and eEF2k+/+ (circles,
n=32) mice. c.40 months old eEF2k-/- mouse.
[0019] FIG. 6. eEF2K-/- mice are resistant to gamma irradiation. a.
The survival of mice after whole-body gamma irradiation. Mice at 8
to 12 weeks of age are exposed to 8 Gy of whole-body
.gamma.-irradiation and survival is monitored daily. Each cohort
contains 10 mice including 5 males and 5 females. b. The
appearances of eEF2K+/+ mice (bottom) and eEF2K-/- mice (top) one
month after 8 Gy of .gamma.-irradiation.
[0020] FIG. 7. eEF2K-/- cells are resistant to apoptosis. a.
Phase-contrast images of cells. MEFs are treated with 1.6 .mu.M of
doxorubicin for 24 hours. b. TUNEL assay of MEFs treated with 1.6
.mu.M of doxorubicin for 12 hours. Apoptotic cells are analyzed by
flow cytometry. c. The effect of introduction of eEF2K cDNA into
eEF2K-/- MEFs on the activation of caspase 3 induced by 0.8 .mu.M
of doxorubicin or 600 .mu.M of H.sub.2O.sub.2. Activation of
caspase 3 is analyzed by western blotting.
[0021] FIG. 8. Phosphorylation of eEF2 occurs in cells undergoing
apoptosis. a. Western blot analysis of phosphorylated eEF2. NIH3T3
cells are exposed to 80 .mu.M of hydrogen peroxide for the
indicated time periods and eEF2 phosphorylation is analyzed by
western blotting using antibodies specific for phosphorylated eEF2.
b. Immunostaining of phosphorylated eEF2 in NIH3T3 cells. Cells are
exposed to 400 .mu.M of H.sub.2O.sub.2 for 3 hours and
phosphorylated eEF2 is detected by immunostaining in both
H.sub.2O.sub.2-treated and untreated (UT) cells. Cells showing the
highest phosphorylated eEF2 levels are indicated by the white
arrowheads. c. HeLa cells are treated with 400 .mu.M of
H.sub.2O.sub.2 for 3 hours and immunostaining is performed. Cells
with condensed chromatin are indicated by gray arrowheads. d,e,f,g.
immunostaining of phosphorylated eEF2 in human lymph nodes and
brain. e. higher magnification of d.
DETAILED DESCRIPTION OF THE INVENTION
[0022] The present invention is based in part on the discovery that
decreasing eEF2 kinase activity in a cell results in an increase in
resistance to damage to the cell, as well as a resistance to
programmed cell death (apoptosis). Without being bound by any
particular theory, it is believed that the increased levels of
protein translation and protein turnover that result from a
decrease in eEF2 kinase activity help protect cells from damage and
subsequent death. The term "protein turnover" is art-recognized and
refers to the coordinated synthesis and degradation of proteins
that occurs in living cells, tissues and organisms.
Cell Death
[0023] Apoptosis is referred to as a process of "programmed cell
death." During normal somatic development, cell populations in
specific organs or tissues may be programmed for death as part of
the developmental progression of tissue remodeling or obsolescence.
See J. J. Cohen, Avd. Immunol. 50:55-85 (1991); M. Baring a,
Science 259:762-3 (1993). Apoptosis is internally triggered by
biochemical or biomolecular mechanisms intrinsic to the cell cycle,
resulting in an activation of endogenous endonucleases (enzymes
that degrade DNA), leading to DNA strand breaks between nucleosomes
and degradation of the genomic DNA by fragmentation. A. H. Wyllie,
Nature 284:555-6 (1980). Apoptosis in mature tissues occurs in
normal processes such as inflammation or rejuvenation. M. Schmied
et al., Am. J. Pathol. 143:446-52 (1993); Abnormal clonal
proliferations in immunologic diseases or malignancies may be
related to a failure of normal apoptosis. J. Marx, Science
259:760-1 (1993).
[0024] The relationship of apoptosis and/or cell damage to the cell
cycle, including checkpoint controls, during cancer chemotherapy is
a subject of interest to oncologists and molecular biologists. See
T. Shimizu et al., Cancer Res. 55:228-231 (1995); O'Connor, supra.
(1992). The expression of p53 in damaged cells is one factor in
determining the course of divergent biochemical pathways, which can
lead to either DNA repair or apoptosis. E Yonish-Rouach et al., Mol
Cell Biol 13:1415-23 (1993); D E Fisher, Cell 78:539-542
(1994).
[0025] In chemotherapy for malignancy, treatments with targeted
cytotoxic effect have involved a number clinical considerations:
they may be used in the primary effort to control cancer (induction
chemotherapy), or as an adjunct to surgery or radiotherapy
(adjuvant chemotherapy). DeVita, supra (1994). Local treatments
have included infusion of a targeted cytotoxic compound into body
cavities to control the spread of malignancies such as breast or
ovarian cancers.
Cell Damage
[0026] Cell damage is caused by a treatment that causes stress to
the cell. In a particularly preferred embodiment. Stress may be
caused by a variety of factors, including increased acidity,
oxidative stress, or exposure of the cell to a compound used for
treatment of a disease state, such as for example, camptothecin
(CPT), doxorubicin (DOX), or taxol. Cell damage may also stem from
ionizing radiation, ultraviolet radiation and free radicals.
Decreasing Kinase Activity
[0027] The present invention relates to a method of reducing damage
to a cell or increasing resistance to damage to a cell, comprising
decreasing eEF2 kinase activity in the cell. The cell may be in
cultures, in a tissue or in a subject in need of treatment. The
subject may be a mammal such as a human.
[0028] The term "inhibition" refers to the reduction or down
regulation of a process or activity that results in the absence or
minimization of that process or activity. The term "inhibit" or
"inhibiting", in relationship to the term "activity" means that an
activity is decreased or prevented in the presence of a compound as
opposed to in the absence of the compound.
[0029] In a preferred embodiment, the decrease in eEF2 kinase
activity, is accomplished by contacting the eEF2 kinase with a
compound that decreases phosphorylation of eEF2 by the eEF2
kinase.
[0030] The term "contact" or "contacting" means bringing at least
two moieties together, whether in an in vitro system or an in vivo
system.
Compounds
[0031] The term "compound" is used herein in the context of a "test
compound" or a "drug candidate compound" described in connection
with the assays of the present invention. As such, these compounds
comprise organic or inorganic compounds, derived synthetically or
from natural sources. The compounds include inorganic or organic
compounds such as polynucleotides, lipids or hormone analogs that
are characterized by relatively low molecular weights. Other
biopolymeric organic test compounds include peptides comprising
from about 2 to about 40 amino acids and larger polypeptides
comprising from about 40 to about 500 amino acids, such as
antibodies or antibody conjugates.
Assays to Identify Compounds
[0032] There are a variety of methods that may be used to identify
compounds capable of inhibition of the activity of eEF2 kinase. The
affinity of the compounds to eEF2 kinase may be determined in an
experiment that detects changed reaction conditions after
phosphorylation of eEF2. eEF2 kinase is incubated with eEF2 and ATP
in an appropriate buffer. The combination of these components
results in the in vitro phosphorylation of eEF2. Sources of
compounds include any commercially available screening library,
peptides in a phage display library or an antibody fragment
library, and compounds that have been demonstrated to have binding
affinity for eEF2 kinase.
[0033] The term "binding affinity" is a property that describes how
strongly two or more compounds associate with each other in a
non-covalent relationship. Binding affinities can be characterized
qualitatively, (such as "strong", "weak", "high", or "low") or
quantitatively (such as measuring the K.sub.D).
[0034] eEF2 kinase can be prepared in a number of ways depending on
whether the assay will be run using cells, cell fractions or
biochemically, on purified protein. eEF2 kinase can be applied as
complete a polypeptides or as a polypeptide fragment, which still
comprises eEF2 kinase catalytic activity.
[0035] The term "assay" means any process used to measure a
specific property of a compound. A "screening assay" means a
process used to characterize or select compounds based upon their
activity from a collection of compounds.
[0036] The term "polypeptide" relates to proteins, proteinaceous
molecules, fractions of proteins peptides and oligopeptides.
[0037] Identification of small molecules inhibiting the activity of
the eEF2 kinase is performed by measuring changes in levels of
phosphorylated eEF2 kinase substrate, which can be a peptide or a
full-length protein, or ATP. A preferred substrate is eEF2. Since
ATP is consumed during the phosphorylation of eEF2 kinase
substrate, its levels correlate with the kinase activity. Measuring
ATP levels via chemiluminescent reactions therefore represents a
method to measure kinase activity in vitro (Perkin Elmer). In a
second type of assay, changes in the levels of phosphorylated eEF2
kinase substrate are detected with phosphospecific agents and are
correlated to eEF2 kinase activity. These levels are detected in
solution or after immobilization of the substrate on a microtiter
plate or other carrier. In solution, the phosphorylated eEF2 kinase
substrate is detected via fluorescence resonance energy transfer
(FRET) between the Eu labeled substrate and an APC labeled
phosphospecific antibody (Perkin Elmer), via fluorescence
polarization (FP) after binding of a phosphospecific antibody to
the fluorescently labeled phosphorylated eEF2 kinase substrate, via
an Amplified Luminescent Proximity Homogeneous Assay (ALPHA) using
the phosphorylated eEF2 kinase substrate and phosphospecific
antibody, both coupled to ALPHA beads (Perkin Elmer) or using the
IMAP binding reagent that specifically detects phosphate groups and
thus alleviates the use of the phosphospecific antibody (Molecular
Devices). Alternatively, the eEF2 kinase substrate is immobilized
directly or by using biotin-streptavidin on a microtiter plate.
After immobilization, the level of phosphorylated eEF2 kinase
substrate is detected using a classic ELISA where binding of the
phosphospecific antibody is either monitored via an enzyme such as
horseradish peroxidase (HRP) or alkaline phosphatase (AP) which are
either directly coupled to the phosphospecific antibody or are
coupled to a secondary antibody. Enzymatic activity correlates to
phosphorylated eEF2 kinase substrate levels. Alternatively, binding
of the Eu-labeled phosphospecific antibody to the immobilized
phosphorylated eEF2 kinase substrate is determined via time
resolved fluorescence energy (TRF) (Perkin Elmer). In addition, the
eEF2 kinase substrate can be coated on FLASH plates (Perkin Elmer)
and phosphorylation of the eEF2 kinase substrate is detected using
.sup.33P labeled ATP or .sup.125I labeled phosphospecific
antibody.
[0038] The term "agent" means any molecule, including polypeptides,
polynucleotides and small molecules.
[0039] Small molecules are randomly screened or are preselected
based upon drug class, (i.e. known kinase inhibitors), or upon
virtual ligand screening (VLS) results. VLS uses virtual docking
technology to test large numbers of small molecules in silico for
their binding to the polypeptide of the invention. Small molecules
are added to the kinase reaction and their effect on levels of
phosphorylated eEF2 is measured with one or more of the
above-described technologies.
[0040] Small molecules that inhibit the kinase activity are
identified and are subsequently tested at different concentrations.
IC.sub.50 values are calculated from these dose response curves.
Strong binders have an IC.sub.50 in the nanomolar and even
picomolar range.
Reduction in eEF2 Gene Expression
[0041] In another preferred embodiment, the present invention
relates to a method of reducing damage to a cell or increasing
resistance to damage to a cell, comprising decreasing eEF2 kinase
activity by reducing the expression of a gene encoding the eEF2
kinase. This reduction in expression can be accomplished by a
variety of methods and in preferred embodiments it is accomplished
by altering the gene such that the gene encodes a dysfunctional or
non-functional eEF2 kinase.
[0042] The term "expression" comprises both endogenous expression
and overexpression by transduction.
[0043] A variety of means are available for altering a gene to
effect expression. In a special embodiment the expression of a gene
encoding the eEF2 kinase is reduced by contacting the gene, or an
mRNA transcribed from the gene, with a compound comprising a
polynucleotide selected from the group consisting of an antisense
oligonucleotide, a ribozyme, a small interfering RNA (siRNA), and a
short hairpin RNA (shRNA). In certain embodiments the compound
comprises a polynucleotide comprising a nucleotide sequence
complementary to a nucleotide sequence encoding a polypeptide
comprising the amino acid sequence of SEQ ID NO: 2, (eEF2 kinase
polypeptide sequence). In a particularly preferred embodiment the
compound comprises a nucleotide sequence complementary to a
nucleotide sequence comprising the nucleotide sequence of SEQ ID
NO: 1 (eEF2 kinase polynucleotide sequence).
[0044] The term "polynucleotide" means a polynucleic acid, in
single or double stranded form, and in the sense or antisense
orientation, complementary polynucleic acids that hybridize to a
particular polynucleic acid under stringent conditions, and
polynucleotides that are homologous in at least about 60 percent of
its base pairs, and more preferably 70 percent of its base pairs
are in common, most preferably 90 percent, and in a special
embodiment 100 percent of its base pairs. The polynucleotides
include polyribonucleic acids, polydeoxyribonucleic acids, and
synthetic analogues thereof. The polynucleotides are described by
sequences that vary in length, that range from about 10 to about
5000 bases, preferably about 100 to about 4000 bases, more
preferably about 250 to about 2500 bases. A preferred
polynucleotide embodiment comprises from about 10 to about 30 bases
in length. A special embodiment of polynucleotide is the
polyribonucleotide of from about 10 to about 22 nucleotides, more
commonly described as small interfering RNAs (siRNAs). Another
special embodiment are nucleic acids with modified backcartilages
such as peptide nucleic acid (PNA), polysiloxane, and
2'-O-(2-methoxy)ethylphosphorothioate, or including non-naturally
occurring nucleic acid residues, or one or more nucleic acid
substituents, such as methyl-, thio-, sulphate, benzoyl-, phenyl-,
amino-, propyl-, chloro-, and methanocarbanucleosides, or a
reporter molecule to facilitate its detection.
[0045] The term "antisense nucleic acid" refers to an
oligonucleotide that has a nucleotide sequence that interacts
through base pairing with a specific complementary nucleic acid
sequence involved in the expression of the target such that the
expression of the gene is reduced. Preferably, the specific nucleic
acid sequence involved in the expression of the gene is a genomic
DNA molecule or mRNA molecule that encodes (a part of) the gene.
This genomic DNA molecule can comprise regulatory regions of the
gene, or the coding sequence for the mature gene.
[0046] The term `complementary to a nucleotide sequence` in the
context of antisense oligonucleotides and methods should be
understood as sufficiently complementary to such a sequence as to
allow hybridization to that sequence in a cell, i.e., under
physiological conditions.
[0047] The term "hybridization" means any process by which a strand
of nucleic acid binds with a complementary strand through base
pairing. The term "hybridization complex" refers to a complex
formed between two nucleic acid sequences by virtue of the
formation of hydrogen bonds between complementary bases. A
hybridization complex may be formed in solution (e.g., C0t or R0t
analysis) or formed between one nucleic acid sequence present in
solution and another nucleic acid sequence immobilized on a solid
support (e.g., paper, membranes, filters, chips, pins or glass
slides, or any other appropriate eEF2 to which cells or their
nucleic acids have been fixed). The term "stringent conditions"
refers to conditions that permit hybridization between
polynucleotides and the claimed polynucleotides. Stringent
conditions can be defined by salt concentration, the concentration
of organic solvent, e.g., formamide, temperature, and other
conditions well known in the art. In particular, reducing the
concentration of salt, increasing the concentration of formamide,
or raising the hybridization temperature can increase
stringency.
Antisense
[0048] The down regulation of gene expression using antisense
nucleic acids can be achieved at the translational or
transcriptional level using an expression-inhibitory agent.
Antisense nucleic acids of the invention are preferably nucleic
acid fragments capable of specifically hybridizing with all or part
of a nucleic acid encoding a eEF2 kinase or the corresponding
messenger gene or mRNA. In addition, antisense nucleic acids may be
designed which decrease expression of the nucleic acid sequence
capable of encoding a eEF2 kinase by inhibiting splicing of its
primary transcript. Any length of antisense sequence is suitable
for practice of the invention so long as it is capable of
down-regulating or blocking expression of a nucleic acid coding for
eEF2 kinase. Preferably, the antisense sequence is at least about
17 nucleotides in length. The preparation and use of antisense
nucleic acids, DNA encoding antisense RNAs and the use of oligo and
genetic antisense is known in the art. \
[0049] The term "expression inhibitory agent" means a
polynucleotide designed to interfere selectively with the
transcription, translation and/or expression of a specific
polypeptide or protein normally expressed within a cell. More
particularly, "expression inhibitory agent" comprises a DNA or RNA
molecule that contains a nucleotide sequence identical to or
complementary to at least about 17 sequential nucleotides within
the polyribonucleotide sequence coding for a specific polypeptide
or protein. Exemplary expression inhibitory molecules include
ribozymes, double stranded siRNA molecules, self-complementary
single-stranded siRNA molecules, genetic antisense constructs, and
synthetic RNA antisense molecules with modified stabilized
backbones.
[0050] One embodiment of expression-inhibitory agent is a nucleic
acid that is antisense to a nucleic acid comprising SEQ ID NO: 1.
For example, an antisense nucleic acid (e.g. DNA) may be introduced
into cells in vitro, or administered to a subject in vivo, as gene
therapy to inhibit cellular expression of nucleic acids comprising
SEQ ID NO: 1. Antisense oligonucleotides preferably comprise a
sequence containing from about 17 to about 100 nucleotides and more
preferably the antisense oligonucleotides comprise from about 18 to
about 30 nucleotides. Antisense nucleic acids may be prepared from
about 10 to about 30 contiguous nucleotides complementary to a
nucleic acid sequence selected from the sequences of SEQ ID NO:
1.
[0051] The antisense nucleic acids are preferably oligonucleotides
and may consist entirely of deoxyribo-nucleotides, modified
deoxyribonucleotides, or some combination of both. The antisense
nucleic acids can be synthetic oligonucleotides. The
oligonucleotides may be chemically modified, if desired, to improve
stability and/or selectivity. Since oligonucleotides are
susceptible to degradation by intracellular nucleases, the
modifications can include, for example, the use of a sulfur group
to replace the free oxygen of the phosphodiester bond. This
modification is called a phosphorothioate linkage. Phosphorothioate
antisense oligonucleotides are water soluble, polyanionic, and
resistant to endogenous nucleases. In addition, when a
phosphorothioate antisense oligonucleotide hybridizes to its mRNA
target, the RN202-315NA duplex activates the endogenous enzyme
ribonuclease (RNase) H, which cleaves the mRNA component of the
hybrid molecule.
[0052] In addition, antisense oligonucleotides with phosphoramidite
and polyamide (peptide) linkages can be synthesized. These
molecules should be very resistant to nuclease degradation.
Furthermore, chemical groups can be added to the 2' carbon of the
sugar moiety and the 5 carbon (C-5) of pyrimidines to enhance
stability and facilitate the binding of the antisense
oligonucleotide to its TARGET site. Modifications may include
2'-deoxy, O-pentoxy, O-propoxy, O-methoxy, fluoro, methoxyethoxy
phosphorothioates, modified bases, as well as other modifications
known to those of skill in the art.
Ribozyme
[0053] Another type of expression-inhibitory agent that reduces the
levels of mRNA is the ribozyme. Ribozymes are catalytic RNA
molecules (RNA enzymes) that have separate catalytic and substrate
binding domains. The substrate binding sequence combines by
nucleotide complementarity and, possibly, non-hydrogen bond
interactions with its mRNA sequence. The catalytic portion cleaves
the mRNA at a specific site. The substrate domain of a ribozyme can
be engineered to direct it to a specified mRNA sequence. The
ribozyme recognizes and then binds eEF2 kinase mRNA through
complementary base pairing. Once it is bound to the correct eEF2
kinase mRNA site, the ribozyme acts enzymatically to cut the eEF2
kinase mRNA. Cleavage of the mRNA by a ribozyme destroys its
ability to direct synthesis of the corresponding polypeptide. Once
the ribozyme has cleaved its eEF2 kinase mRNA sequence, it is
released and can repeatedly bind and cleave at other mRNAs.
Ribozyme forms include a hammerhead motif, a hairpin motif, a
hepatitis delta virus, group I intron or RNaseP RNA (in association
with an RNA guide sequence) motif or Neurospora VS RNA motif.
Ribozymes possessing a hammerhead or hairpin structure are readily
prepared since these catalytic RNA molecules can be expressed
within cells from eukaryotic promoters (Chen, et al. (1992) Nucleic
Acids Res. 20:4581-9). A ribozyme of the present invention can be
expressed in eukaryotic cells from the appropriate DNA vector.
[0054] If desired, the activity of the ribozyme may be augmented by
its release from the primary transcript by a second ribozyme
(Ventura, et al. (1993) Nucleic Acids Res. 21:3249-55).
[0055] The term "vectors" relates to plasmids as well as to viral
vectors, such as recombinant viruses, or the nucleic acid encoding
the recombinant virus.
[0056] Ribozymes may be chemically synthesized by combining an
oligodeoxyribonucleotide with a ribozyme catalytic domain (20
nucleotides) flanked by sequences that hybridize to the eEF2 kinase
mRNA after transcription. The oligodeoxyribonucleotide is amplified
by using the substrate binding sequences as primers. The
amplification product is cloned into a eukaryotic expression
vector.
[0057] Ribozymes are expressed from transcription units inserted
into DNA, RNA, or viral vectors. Transcription of the ribozyme
sequences are driven from a promoter for eukaryotic RNA polymerase
I (pol (I), RNA polymerase II (pol II), or RNA polymerase III (pol
III). Transcripts from pol II or pol III promoters will be
expressed at high levels in all cells; the levels of a given pol II
promoter in a given cell type will depend on nearby gene regulatory
sequences. Prokaryotic RNA polymerase promoters are also used,
providing that the prokaryotic RNA polymerase enzyme is expressed
in the appropriate cells (Gao and Huang, (1993) Nucleic Acids Res.
21:2867-72). It has been demonstrated that ribozymes expressed from
these promoters can function in mammalian cells (Kashani-Sabet, et
al. (1992) Antisense Res. Dev. 2:3-15).
siRNA
[0058] A particularly preferred inhibitory agent is a small
interfering RNA (siRNA). siRNA, preferably short hairpin RNA
(shRNA), mediate the post-transcriptional process of gene silencing
by double stranded RNA (dsRNA) that is homologous in sequence to
the silenced RNA. siRNA according to the present invention
comprises a sense strand of 17-25 nucleotides complementary or
homologous to a contiguous 17-25 nucleotide sequence selected from
the group of sequences encoding SEQ ID NO: 2, preferably from SEQ
ID NO: 1, and an antisense strand of 17-23 nucleotides
complementary to the sense strand. The most preferred siRNA
comprises sense and anti-sense strands that are 100 percent
complementary to each other and the eEF2 kinase polynucleotide
sequence. Preferably the siRNA further comprises a loop region
linking the sense and the antisense strand. A self-complementing
single stranded siRNA molecule polynucleotide according to the
present invention comprises a sense portion and an antisense
portion connected by a loop region linker. The loop can be any
length but is preferably 4-30 nucleotides long. Self-complementary
single stranded siRNAs form hairpin loops and are more stable than
ordinary dsRNA. In addition, they are more easily produced from
vectors.
[0059] Analogous to antisense RNA, the siRNA can be modified to
confirm resistance to nucleolytic degradation, or to enhance
activity, or to enhance cellular distribution, or to enhance
cellular uptake, such modifications may consist of modified
internucleoside linkages, modified nucleic acid bases, modified
sugars and/or chemical linkage the siRNA to one or more moieties or
conjugates.
[0060] The present invention also relates to compositions, and
methods using said compositions, comprising a DNA expression vector
capable of expressing a polynucleotide capable of increasing
resistance to cell damage and is described hereinabove as an
expression inhibition agent.
Intracellular Binding Protein
[0061] A special aspect of these compositions and methods relates
to the down-regulation or blocking of the expression of a eEF2
kinase by the induced expression of a polynucleotide encoding an
intracellular binding protein that is capable of selectively
interacting with the eEF2 kinase polypeptide. An intracellular
binding protein includes any protein capable of selectively
interacting, or binding, with the polypeptide in the cell in which
it is expressed and neutralizing the function of the polypeptide.
Preferably, the intracellular binding protein is a neutralizing
antibody or a fragment of a neutralizing antibody having binding
affinity to an epitope of the eEF2 kinase of SEQ ID NO: 2. More
preferably, the intracellular binding protein is a single chain
antibody.
[0062] The term "binding affinity" is a property that describes how
strongly two or more compounds associate with each other in a
non-covalent relationship. Binding affinities can be characterized
qualitatively, (such as "strong", "weak", "high", or "low") or
quantitatively (such as measuring the K.sub.D).
[0063] A special embodiment of this composition comprises the
expression-inhibiting agent selected from the group consisting of
antisense RNA, antisense oligodeoxynucleotide (ODN), a ribozyme
that cleaves the polyribonucleotide coding for SEQ ID NO: 2, and a
small interfering RNA (siRNA) that is sufficiently homologous to a
portion of the polyribonucleotide coding for SEQ ID NO: 2, such
that the siRNA interferes with the translation of the eEF2 kinase
polyribonucleotide to the eEF2 kinase polypeptide.
[0064] The polynucleotide expressing the expression-inhibiting
agent is preferably included within a vector. The polynucleic acid
is operably linked to signals enabling expression of the nucleic
acid sequence and is introduced into a cell utilizing, preferably,
recombinant vector constructs, which will express the antisense
nucleic acid once the vector is introduced into the cell. A variety
of viral-based systems are available, including adenoviral,
retroviral, adeno-associated viral, lentiviral, herpes simplex
viral or a sendaviral vector systems, and all may be used to
introduce and express polynucleotide sequence for the
expression-inhibiting agents in eEF2 kinase-expressing cells.
[0065] The term "operably linked" or "operably inserted" means that
the regulatory sequences necessary for expression of the coding
sequence are placed in a nucleic acid molecule in the appropriate
positions relative to the coding sequence so as to enable
expression of the coding sequence. This same definition is
sometimes applied to the arrangement other transcription control
elements (e.g. enhancers) in an expression vector. Transcriptional
and translational control sequences are DNA expression regulatory
sequences, such as promoters, enhancers, polyadenylation signals,
terminators, and the like, that provide for the expression of a
coding sequence in a host cell.
[0066] The terms "promoter", "promoter region" or "promoter
sequence" refer generally to transcriptional regulatory regions of
a gene, which may be found at the 5' or 3'side of the coding
region, or within the coding region, or within introns. Promoters
that may be used in the expression vectors of the present invention
include both constitutive promoters and regulated (inducible)
promoters.
[0067] Typically, a promoter is a DNA regulatory region capable of
binding RNA polymerase in a cell and initiating transcription of a
downstream (3'direction) coding sequence. The typical 5'promoter
sequence is bounded at its 3'terminus by the transcription
initiation site and extends upstream (5'direction) to include the
minimum number of bases or elements necessary to initiate
transcription at levels detectable above background. Within the
promoter sequence is a transcription initiation site (conveniently
defined by mapping with nuclease S1), as well as protein binding
domains (consensus sequences) responsible for the binding of RNA
polymerase.
[0068] In a preferred embodiment, the viral element is derived from
an adenovirus. Other embodiments of the present invention use
retroviral vector systems which can be constructed from different
types of retrovirus, such as, MoMuLV ("murine Moloney leukemia
virus" MSV ("murine Moloney sarcoma virus"), HaSV ("Harvey sarcoma
virus"); SNV ("spleen necrosis virus"); RSV ("Rous sarcoma virus")
and Friend virus. Lentiviral vector systems may also be used in the
practice of the present invention. In other embodiments of the
present invention, adeno-associated viruses ("AAV") are
utilized.
[0069] Preferably, the viral vectors used in the methods of the
present invention are replication defective. Such replication
defective vectors will usually pack at least one region that is
necessary for the replication of the virus in the infected cell.
These regions can either be eliminated (in whole or in part), or be
rendered non-functional by any technique known to a person skilled
in the art. These techniques include the total removal,
substitution, partial deletion or addition of one or more bases to
an essential (for replication) region. Such techniques may be
performed in vitro (on the isolated DNA) or in situ, using the
techniques of genetic manipulation or by treatment with mutagenic
agents. Preferably, the replication defective virus retains the
sequences of its genome, which are necessary for encapsidating, the
viral particles.
[0070] In the vector construction, the polynucleotide agents of the
present invention may be linked to one or more regulatory regions.
Selection of the appropriate regulatory region or regions is a
routine matter, within the level of ordinary skill in the art.
Regulatory regions include promoters, and may include enhancers,
suppressors, etc.
[0071] Additional vector systems include the non-viral systems that
facilitate introduction of polynucleotide agents into a patient.
For example, a DNA vector encoding a desired sequence can be
introduced in vivo by lipofection. Synthetic cationic lipids
designed to limit the difficulties encountered with
liposome-mediated transfection can be used to prepare liposomes for
in vivo transfection of a gene encoding a marker (Feigner, et. al.
(1987) Proc. Natl. Acad Sci. USA 84:7413-7); see Mackey, et al.
(1988) Proc. Natl. Acad. Sci. USA 85:8027-31; Ulmer, et al. (1993)
Science 259:1745-8). The use of cationic lipids may promote
encapsulation of negatively charged nucleic acids, and also promote
fusion with negatively charged cell membranes (Feigner and Ringold,
(1989) Nature 337:387-8). Particularly useful lipid compounds and
compositions for transfer of nucleic acids are described in
International Patent Publications WO 95/18863 and WO 96/17823, and
in U.S. Pat. No. 5,459,127. The use of lipofection to introduce
exogenous genes into the specific organs in vivo has certain
practical advantages and directing transfection to particular cell
types would be particularly advantageous in a tissue with cellular
heterogeneity, for example, pancreas, liver, kidney, and the brain.
Lipids may be chemically coupled to other molecules for the purpose
of targeting. Targeted peptides, e.g., hormones or
neurotransmitters, and proteins for example, antibodies, or
non-peptide molecules could be coupled to liposomes chemically.
Other molecules are also useful for facilitating transfection of a
nucleic acid in vivo, for example, a cationic oligopeptide (e.g.,
International Patent Publication WO 95/21931), peptides derived
from DNA binding proteins (e.g., International Patent Publication
WO 96/25508), or a cationic polymer (e.g., International Patent
Publication WO 95/21931).
[0072] It is also possible to introduce a DNA vector in vivo as a
naked DNA plasmid (see U.S. Pat. Nos. 5,693,622, 5,589,466 and
5,580,859). Naked DNA vectors for therapeutic purposes can be
introduced into the desired host cells by methods known in the art,
e.g., transfection, electroporation, microinjection, transduction,
cell fusion, DEAE dextran, calcium phosphate precipitation, use of
a gene gun, or use of a DNA vector transporter (see, e.g., Wilson,
et al. (1992) J. Biol. Chem. 267:963-7; Wu and Wu, (1988) J. Biol.
Chem. 263:14621-4; Hartmut, et al. Canadian Patent Application No.
2,012,311, filed Mar. 15, 1990; Williams, et al (1991). Proc. Natl.
Acad. Sci. USA 88:2726-30). Receptor-mediated DNA delivery
approaches can also be used (Curiel, et al. (1992) Hum. Gene Ther.
3:147-54; Wu and Wu, (1987) J. Biol. Chem. 262:4429-32).
Methods of Treatment
[0073] In another aspect, the invention relates to a method of
treating and/or preventing a disease characterized by an increase
in eEF2 kinase activity in a patient by administering to the
patient a therapeutically effective amount of a composition
comprising a compound that decreases phosphorylation of eEF2 kinase
substrate by eEF2 kinase. The term "condition" or "disease" means
the overt presentation of symptoms (i.e., illness) or the
manifestation of abnormal clinical indicators (e.g., biochemical
indicators). Alternatively, the term "disease" refers to a genetic
or environmental risk of or propensity for developing such symptoms
or abnormal clinical indicators. A variety of disease states may be
treated utilizing the methods and compounds of the present
invention. In preferred embodiments the disease state is selected
from the group consisting of hypoxia, anoxia, ischemia, stroke, and
neurogenerative diseases such as Parkinson's or Alzheimer's
disease.
[0074] In yet another aspect, the invention relates to a method of
protecting a cell population in a patient from a potential source
of cell damage selected from chemotherapy agents, ionizing
radiation, ultraviolet radiation, and free radicals by
administering to the patient a therapeutically effective amount of
a composition including a compound that decreases phosphorylation
of eEF2 kinase substrate by eEF2 kinase. In particular, the
invention relates to a method of protecting normal tissues during
chemotherapy of cancer cells by administering to the patient a
therapeutically effective amount of a composition including a
compound that decreases phosphorylation of eEF2 kinase substrate by
eEF2 kinase. In one aspect of the invention, the compound includes
a polynucleotide having a nucleotide sequence complementary to a
nucleotide sequence encoding a polypeptide comprising the amino
acid sequence of SEQ ID NO: 2. In another aspect, the ionizing
radiation includes gamma radiation.
[0075] In another aspect, the present invention relates to a
knockout mouse, wherein the knockout mouse comprises a disruption
in an eEF2 kinase gene. In a preferred embodiment, the mouse is
heterozygous for the disruption in the eEF2 kinase gene. In an
especially preferred embodiment, the mouse is homozygous for the
disruption in the eEF2 kinase gene. In a preferred embodiment, the
disruption occurs in a region of the eEF2 kinase gene which encodes
a catalytic domain of the eEF2 kinase polypeptide, preferably the
region comprises exon 7 or 8 of the eEF2 kinase gene. In an
especially preferred embodiment, the mouse exhibits a phenotype
selected from the group consisting of extension of life span and
resistance to stress-induced cell damage.
[0076] In another aspect, the invention relates to an eEF2 kinase
knockout construct, comprising a portion of an eEF2 kinase gene,
wherein a portion of the eEF2 kinase gene is replaced by a
selectable marker. In a preferred embodiment the selectable marker
is a gene which encodes for a polypeptide selected from the group
consisting of thymidine kinase, neomycin phosphotransferase and
hygromycin B phosphotransferase. In an especially preferred
embodiment the portion of the eEF2 kinase gene which is replaced
comprises exon 7.
[0077] In another aspect, the invention relates to a method of
producing a mouse with a targeted disruption in an eEF2 kinase
gene. The mouse is obtained by transfecting a population of
embryonic stem cells with a knockout construct in which comprising
a portion of the eEF2 kinase gene with a portion of the eEF2 kinase
gene replaced by a marker; selecting a transfected embryonic stem
cell which expresses the marker; introducing the transfected ES
cell into an embryo of an ancestor of the mouse allowing the embryo
to develop to term to produce a chimeric mouse with the knockout
construct in its germline; and breeding the chimeric mammal, to
produce a heterozygous mouse with a targeted disruption in the eEF2
kinase gene.
[0078] An eEF2 kinase knockout construct is typically prepared by
isolating a portion of the genomic or cDNA eEF2 kinase nucleotide
sequence (usually encoding at least one exon and one intron), and
inserting a marker sequence into the eEF2 kinase sequence. The eEF2
kinase gene or gene fragment to be used in preparing this construct
may be obtained in a variety of ways. Generally, the eEF2 kinase
DNA molecule will be at least about 1 kilobase (kb) in length, and
preferably will be 3-4 kb in length, thereby providing sufficient
complementary sequence for recognition with chromosomal DNA (i.e.,
homologous recombination) when the knockout construct is introduced
into the genomic DNA of the embryonic stem (ES) cell (discussed
below).
[0079] A naturally occurring genomic eEF2 kinase fragment or cDNA
molecule to be used in preparing the knockout construct can be
obtained using methods well known in the art such as those
described by Sambrook et al. (Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y. (1989)). Such methods include, for example, PCR amplification
of a particular DNA sequence using oligonucleotide primers, or
screening a genomic library prepared from cells or tissues that
contain the eEF2 kinase gene with a cDNA probe encoding at least a
portion of the same or a highly homologous eEF2 kinase gene in
order to obtain at least a portion of the eEF2 kinase genomic
sequence. Alternatively, if a cDNA sequence is to be used in a
knockout construct, the cDNA may be obtained by screening a cDNA
library (preferably one prepared from tissues or that express eEF2
kinase, where the tissues or cells are derived from the same or a
similar species of mammal as that to be rendered the knockout
mammal) with oligonucleotide probes, homologous cDNA probes, or
antibodies (where the library is cloned into an expression vector).
If a promoter sequence is to be used in the knockout construct,
synthetic DNA probes or primers can be designed for screening a
genomic library or for amplification using PCR, respectively.
[0080] The eEF2 kinase genomic DNA fragment or eEF2 kinase cDNA
molecule prepared for use in the knockout construct must be
generated in sufficient quantity for genetic manipulation.
Amplification may be conducted by 1) placing the fragment into a
suitable vector and transforming bacterial or other cells that can
rapidly amplify the vector, 2) by PCR amplification, 3) by
synthesis with a DNA synthesizer, or 4) by other suitable
methods.
[0081] The eEF2 kinase genomic DNA fragment, cDNA molecule, or PCR
fragment to be used in making the eEF2 kinase knockout construct
can be digested with one or more restriction enzymes selected to
cut at a location(s) such that a second DNA molecule encoding a
marker gene can be inserted in the proper position within the eEF2
kinase genomic DNA fragment, cDNA molecule, or PCR fragment to be
used in the construct. The proper position for marker gene
insertion is one that will serve to decrease or prevent
transcription and/or expression of the full length endogenous eEF2
kinase gene. This position will depend on various factors such as
the available restriction sites in the sequence to be cut, whether
an exon sequence or a promoter sequence, or both is (are) to be
interrupted, and whether several isoforms of eEF2 kinase exist in
the mammal (due to alternative splicing) and only one such isoform
is to be disrupted. Preferably, the enzyme(s) selected for cutting
the eEF2 kinase genomic DNA, cDNA molecule, or PCR fragment will
generate a longer arm and a shorter arm, where the shorter arm is
at least about 300 base pairs (bp). In some cases, it will be
desirable to actually delete a portion or even all of one or more
introns or exons of this native genomic or cDNA molecule. In these
cases, the eEF2 kinase genomic DNA, cDNA molecule, or PCR fragment
can be cut with appropriate restriction endonucleases such that a
fragment of the proper size and proper location can be removed.
[0082] The marker gene used in the knockout construct can be any
nucleic acid molecule that is detectable and/or assayable after it
has been incorporated into the genomic DNA of the ES cell, and
ultimately the knockout mammal, however typically it is an
antibiotic resistance gene or other gene whose expression or
presence in the genome can easily be detected. Preferably, the
marker gene encodes a polypeptide that does not naturally occur in
the mammal. The marker gene is usually operably linked to its own
promoter or to another strong promoter such as the thymidine kinase
(TK) promoter or the phosphoglycerol kinase (PGK) promoter from any
source that will be active or can easily be activated in the cell
into which it is inserted; however, the marker gene need not have
its own promoter attached, as it may be transcribed using the
promoter of the gene to be knocked out. In addition, the marker
gene will normally have a polyA sequence attached to its 3' end;
this sequence serves to terminate transcription of the marker gene.
Preferred marker genes are any antibiotic resistance gene such as
neo (the neomycin resistance gene) and beta-gal
(beta-galactosidase).
[0083] After the eEF2 kinase genomic DNA fragment, cDNA molecule,
or PCR fragment has been digested with the appropriate restriction
enzyme(s), the marker gene molecule can be ligated with the native
genomic DNA or cDNA molecule using methods well known to the
skilled artisan and described in Sambrook et al., supra. In some
cases, it will be preferable to insert the marker sequence in the
reverse or antisense orientation with respect to the eEF2 kinase
nucleic acid sequence; this reverse insertion is preferred where
the marker gene is operably linked to a particularly strong
promoter.
[0084] The ends of the DNA molecules to be ligated must be
compatible; this can be achieved by either cutting all fragments
with those enzymes that generate compatible ends, or by blunting
the ends prior to ligation. Blunting can be done using methods well
known in the art, such as for example by the use of Klenow fragment
(DNA polymerase I) to fill in sticky ends. After ligation, the
ligated constructs can be screened by selective restriction
endonuclease digestion to determine which constructs contain the
marker sequence in the desired orientation.
[0085] The ligated DNA knockout construct may be transfected
directly into embryonic stem cells (discussed below), or it may
first be placed into a suitable vector for amplification prior to
insertion. Preferred vectors are those that are rapidly amplified
in bacterial cells such as the pBluescript II SK vector
(Stratagene, San Diego, Calif.) or pGEM7 (Promega Corp., Madison,
Wis.).
[0086] The eEF2 kinase knockout construct is typically transfected
into stem cells derived from an embryo (embryonic stem cells, or
"ES cells"). ES cells are undifferentiated cells that are capable
of taking up extra-chromosomal DNA and incorporating it into their
chromosomal DNA. Generally, the ES cells used to produce the
knockout mammal will be of the same species as the knockout mammal
to be generated. Thus for example, mouse embryonic stem cells will
usually be used for generation of knockout mice.
[0087] The embryonic stem cell line used is typically selected for
its ability to integrate into and become part of the germ line of a
developing embryo so as to create germ line transmission of the
knockout construct. Thus, any ES cell line that is believed to have
this capability is suitable for use herein. Preferred ES cell lines
for generating knockout mice are murine cell lines D3 and E14
(American Type Culture Collection, 12301 Parklawn Drive, Rockville,
Md. 20852-1776 USA, catalog nos. CRL 1934 and CRL 1821,
respectively), or RW4 (Genome Systems, Inc., 8620 Pennell Drive,
St. Louis, Mich. 63114 USA, catalog No. ESVJ-1182). The cells are
cultured and prepared for DNA insertion using methods well known to
the skilled artisan such as those set forth by Robertson (in:
Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.
J. Robertson, ed. IRL Press, Washington, D.C. (1987)), by Bradley
et al. (Current Topics in Devel. Biol., 20:357-371 (1986)) and by
Hogan et al. (Manipulating the Mouse Embryo: A Laboratory Manual,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1986)).
[0088] Insertion (also termed "transfection") of the knockout
construct into the ES cells can be accomplished using a variety of
methods well known in the art including for example,
electroporation, microinjection, and calcium phosphate treatment
(see Lovell-Badge, in Robertson, ed., supra). A preferred method of
insertion is electroporation.
[0089] The eEF2 kinase knockout construct DNA molecules to be
transfected into the cells can first be linearized if the knockout
construct has previously been inserted into a circular vector.
Linearization can be accomplished by digesting the DNA with a
suitable restriction endonuclease selected to cut only within the
vector sequence and not within the knockout construct sequence.
[0090] The isolated eEF2 kinase knockout construct DNA can be added
to the ES cells under appropriate conditions for the insertion
method chosen. Where more than one construct is to be introduced
into the ES cells, the DNA molecules encoding each construct can be
introduced simultaneously or sequentially. Optionally, homozygous
eEF2 kinase knockout ES cells may be generated by adding excessive
eEF2 kinase knockout construct DNA to the cells, or by conducting
successive rounds of transfection in an attempt to achieve
homologous recombination of the knockout construct on both
endogenous eEF2 kinase alleles.
[0091] If the ES cells are to be electroporated, the ES cells and
knockout construct DNA are exposed to an electric pulse using an
electroporation machine and following the manufacturer's guidelines
for use. After electroporation, the cells are typically allowed to
recover under suitable incubation conditions. The cells are then
screened for the presence of the knockout construct.
[0092] Screening the ES cells can be accomplished using a variety
of methods, but typically, one screens for the presence of the
marker sequence portion of the knockout construct. Where the marker
gene is an antibiotic resistance gene, the cells can be cultured in
the presence of an otherwise lethal concentration of antibiotic.
Those cells that survive have presumably integrated the knockout
construct. If the marker gene is other than an antibiotic
resistance gene, a Southern blot of the ES cell genomic DNA can be
probed with a sequence of DNA designed to hybridize only to the
marker sequence. If the marker gene is a gene that encodes an
enzyme whose activity can be detected (e.g., beta-galactosidase),
the enzyme substrate can be added to the cells under suitable
conditions, and the enzymatic activity of the marker gene can be
analyzed.
[0093] The knockout construct may integrate into several locations
in the ES cell genome, and may integrate into a different location
in each cell's genome, due to the occurrence of random insertion
events; the desired location of insertion is within the eEF2 kinase
endogenous gene sequence. Typically, less than about 1-10 percent
of the ES cells that take up the knockout construct will actually
integrate the knockout construct in the desired location. To
identify those cells with proper integration of the knockout
construct, chromosomal DNA can be extracted from the cells using
standard methods such as those described by Sambrook et al., supra.
This DNA can then be probed on a Southern blot with a probe or
probes designed to hybridize to the knockout construct DNA digested
with (a) particular restriction enzyme(s). Alternatively, or
additionally, a specific genomic DNA sequence can be amplified by
PCR with probes specifically designed to amplify that DNA sequence
such that only those cells containing the knockout construct in the
proper position will generate DNA fragments of the proper size.
[0094] Accordingly, the present invention also relates to an
isolated cell, wherein the cell contains a disruption in the eEF2
kinase gene. The cell can be any type of cell, including cells
isolated from a non-human animal that is homozygous or heterozygous
for the disruption to the gene, for example, mouse embryo
fibroblasts (MEFs). In preferred embodiments the cell is an
undifferentiated cell. In particularly preferred embodiments the
undifferentiated cell is selected from the group consisting of a
stem cell, an embryonic stem cell, an oocyte and an embryonic cell.
In especially preferred embodiment, the cell comprises a disruption
of the eEF2 kinase gene which encodes a catalytic domain of the
eEF2 kinase polypeptide. In an especially preferred embodiment, the
disruption comprises a portion of an eEF2 kinase gene, wherein a
portion of the eEF2 kinase gene is replaced by a selectable marker.
In a preferred embodiment the selectable marker is a gene which
encodes for a polypeptide selected from the group consisting of
thymidine kinase, neomycin phosphotransferase and hygromycin B
phosphotransferase. In an especially preferred embodiment the
portion of the eEF2 kinase gene which is replaced comprises exon
7.
[0095] After suitable ES cells containing the knockout construct in
the proper location have been identified, the cells can be
incorporated into an embryo. Incorporation may be accomplished in a
variety of ways. A preferred method of incorporation of ES cells is
by microinjection into an embryo that is at the blastocyst stage of
development. For microinjection, about 10-30 cells are collected
into a micropipet and injected into a blastocyst to integrate the
ES cell into the developing blastocyst.
[0096] The suitable stage of development for the blastocyst is
species dependent, however for mice it is about 3.5 days. The
blastocysts can be obtained by perfusing the uterus of pregnant
females. Suitable methods for accomplishing this are known to the
skilled artisan, and are set forth for example by Bradley (in
Robertson, ed., supra).
[0097] While any blastocyst of the right age/stage of development
is suitable for use, preferred blastocysts are male and have genes
coding for a coat color or other phenotypic marker that is
different from the coat color or other phenotypic marker encoded by
the ES cell genes. In this way, the offspring can be screened
easily for the presence of the knockout construct by looking for
mosaic coat color or other phenotypic marker (indicating that the
ES cell is incorporated into the developing embryo). Thus, for
example, if the ES cell line carries the genes for white fur, the
embryo selected will preferably carry genes for black or brown
fur.
[0098] An alternate method of preparing an embryo containing ES
cells that possess the knockout construct is to generate
"aggregation chimeras". A morula of the proper developmental stage
(about 21/2 days old for mice) is isolated. The zona pellucida can
be removed by treating the morula with a solution of mild acid for
about 30 seconds, thereby exposing the "clump" of cells that
comprise the morula. Certain types of ES cells such as the R1 cell
line for mice can then be co-cultured with the morula cells,
forming an aggregation chimera embryo of morula and ES cells.
[0099] A refinement of the aggregation chimera embryo method can be
used to generate an embryo comprised of essentially only those ES
cells containing the knockout construct. In this technique, a very
early stage zygote (e.g., a two-cell stage zygote for mice) is
given a mild electric shock. This shock serves to fuse the nuclei
of the cells in the zygote thereby generating a single nucleus that
has two-fold (or more) the DNA of a naturally occurring zygote of
the same developmental stage. These zygotic cells are excluded from
the developing embryo proper, and contribute only to forming
accessory embryonic structures such as the extra-embryonic
membrane. Therefore, when ES cells are co-cultured with the zygotic
cells, the developing embryo is comprised exclusively of ES
cells.
[0100] After the ES cells have been incorporated, the aggregation
chimera or transfected embryo can be implanted into the uterus of a
pseudopregnant foster mother. While any foster mother may be used,
preferred foster mothers are typically selected for their ability
to breed and reproduce well, and for their ability to care for
their young. Such foster mothers are typically prepared by mating
with vasectomized males of the same species. The pseudopregnant
stage of the foster mother is important for successful
implantation, and it is species dependent. For mice, this stage is
about 2-3 days pseudopregnant.
[0101] Offspring that are born to the foster mother may be screened
initially for mosaic coat color or other phenotype marker where the
phenotype selection strategy (such as coat color, as described
above) has been employed. In addition, or as an alternative,
chromosomal DNA obtained from tail tissue of the offspring may be
screened for the presence of the knockout construct using Southern
blots and/or PCR as described above. The offspring that are
positive for the eEF2 kinase knockout construct will typically be
heterozygous, although some homozygous knockouts may exist, and can
typically be detected by visually quantifying the amount of probe
that hybridizes to the Southern blots.
[0102] If homozygous knockout mammals are desired, they can be
prepared by crossing those heterozygous offspring believed to carry
the knockout construct in their germ line to each other; such
crosses may generate homozygous knockout animals. If it is unclear
whether the offspring will have germ line transmission, they can be
crossed with a parental or other strain and the offspring screened
for heterozygosity. Homozygotes may be identified by Southern
blotting of equivalent amounts of genomic DNA from mammals that are
the product of this cross, as well as mammals of the same species
that are known heterozygotes, and wild-type mammals. Probes to
screen the Southern blots for the presence of the knockout
construct in the genomic DNA can be designed as set forth
above.
[0103] Other means of identifying and characterizing the knockout
offspring are also available. For example, Northern blots can be
used to probe mRNA obtained from various tissues of the offspring
for the presence or absence of transcripts encoding either the gene
knocked out, the marker gene, or both. In addition, Western blots
can be used to assess the level of expression of the gene knocked
out in various tissues of these offspring by probing the Western
blot with an antibody against the protein encoded by the gene
knocked out, or an antibody against the marker gene product, where
this gene is expressed. Finally, in situ analysis (such as fixing
the cells and labeling with antibody) and/or FACS (fluorescence
activated cell sorting) analysis of various cells from the
offspring can be conducted using suitable antibodies to look for
the presence or absence of the knockout construct gene product.
[0104] Both the heterozygous and homozygous eEF2 kinase knockout
mammals of this invention will have a variety of uses, since eEF2
kinase has been implicated in regulation increased life span and
increased resistance to cell damage.
[0105] A functional knockout may also be achieved by the
introduction of an anti-sense construct that blocks expression of
eEF2 kinase.
Compositions
[0106] The present invention also provides biologically compatible,
cell damage-inhibiting compositions comprising an effective amount
of one or more compounds identified as eEF2 kinase inhibitors,
and/or the expression-inhibiting agents as described hereinabove.
In certain aspects, the invention relates to a pharmaceutical
composition for the treatment or prevention of a condition
involving cell damage or a susceptibility to cell damage,
comprising a therapeutically effective amount of a compound that
decreases phosphorylation of eEF2 by eEF2 kinase. In another
aspect, the compound includes a polynucleotide comprising a
nucleotide sequence complementary to a nucleotide sequence encoding
a polypeptide comprising the amino acid sequence of SEQ ID NO:
2.
[0107] The term "effective amount" or "therapeutically effective
amount" means that amount of a compound or agent that will elicit
the biological or medical response of a subject that is being
sought by a medical doctor or other clinician.
[0108] A biologically compatible composition is a composition, that
may be solid, liquid, gel, or other form, in which the compound,
polynucleotide, vector, and antibody of the invention is maintained
in an active form, e.g., in a form able to effect a biological
activity. For example, a compound of the invention would have
inverse agonist or antagonist activity on the eEF2 kinase; a
nucleic acid would be able to replicate, translate a message, or
hybridize to a complementary mRNA of a eEF2 kinase; a vector would
be able to transfect a eEF2 kinase cell and expression the
antisense, antibody, ribozyme or siRNA as described hereinabove; an
antibody would bind a eEF2 kinase polypeptide domain.
[0109] A preferred biologically compatible composition is an
aqueous solution that is buffered using, e.g., Tris, phosphate, or
HEPES buffer, containing salt ions. Usually the concentration of
salt ions will be similar to physiological levels. Biologically
compatible solutions may include stabilizing agents and
preservatives. In a more preferred embodiment, the biocompatible
composition is a pharmaceutically acceptable composition. Such
compositions can be formulated for administration by topical, oral,
parenteral, intranasal, subcutaneous, and intraocular, routes.
Parenteral administration is meant to include intravenous
injection, intramuscular injection, intraarterial injection or
infusion techniques. The composition may be administered
parenterally in dosage unit formulations containing standard,
well-known non-toxic physiologically acceptable carriers, adjuvants
and vehicles as desired.
[0110] The term "carrier" means a non-toxic material used in the
formulation of pharmaceutical compositions to provide a medium,
bulk and/or useable form to a pharmaceutical composition. A carrier
may comprise one or more of such materials such as an excipient,
stabilizer, or an aqueous pH buffered solution. Examples of
physiologically acceptable carriers include aqueous or solid buffer
ingredients including phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid; low molecular weight (less
than about 10 residues) polypeptide; proteins, such as serum
albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, arginine or lysine; monosaccharides, disaccharides, and
other carbohydrates including glucose, mannose, or dextrins;
chelating agents such as EDTA; sugar alcohols such as mannitol or
sorbitol; salt-forming counterions such as sodium; and/or nonionic
surfactants such as TWEEN.TM., polyethylene glycol (PEG), and
PLURONICS.TM..
[0111] A particularly preferred embodiment of the present
composition invention is a cell damage-inhibiting pharmaceutical
composition comprising a therapeutically effective amount of an
expression-inhibiting agent as described hereinabove, in admixture
with a pharmaceutically acceptable carrier. Another preferred
embodiment is a pharmaceutical composition for the treatment or
prevention of a condition related to cell damage, or a
susceptibility to the condition, comprising an effective cell
damage-inhibiting amount of a eEF2 kinase antagonist or inverse
agonist, its pharmaceutically acceptable salts, hydrates, solvates,
or prodrugs thereof in admixture with a pharmaceutically acceptable
carrier.
[0112] The term "pharmaceutically acceptable salts" refers to the
non-toxic, inorganic and organic acid addition salts, and base
addition salts, of compounds of the present invention. These salts
can be prepared in situ during the final isolation and purification
of compounds useful in the present invention.
[0113] The term "solvate" means a physical association of a
compound useful in this invention with one or more solvent
molecules. This physical association includes hydrogen bonding. In
certain instances the solvate will be capable of isolation, for
example when one or more solvent molecules are incorporated in the
crystal lattice of the crystalline solid. "Solvate" encompasses
both solution-phase and isolable solvates. Representative solvates
include hydrates, ethanolates and methanolates.
[0114] Pharmaceutical compositions for oral administration can be
formulated using pharmaceutically acceptable carriers well known in
the art in dosages suitable for oral administration. Such carriers
enable the pharmaceutical compositions to be formulated as tablets,
pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions, and the like, for ingestion by the patient.
Pharmaceutical compositions for oral use can be prepared by
combining active compounds with solid excipient, optionally
grinding a resulting mixture, and processing the mixture of
granules, after adding suitable auxiliaries, if desired, to obtain
tablets or dragee cores. Suitable excipients are carbohydrate or
protein fillers, such as sugars, including lactose, sucrose,
mannitol, or sorbitol; starch from corn, wheat, rice, potato, or
other plants; cellulose, such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxymethyl-cellulose;
gums including arabic and tragacanth; and proteins such as gelatin
and collagen. If desired, disintegrating or solubilizing agents may
be added, such as the cross-linked polyvinyl pyrrolidone, agar,
alginic acid, or a salt thereof, such as sodium alginate. Dragee
cores may be used in conjunction with suitable coatings, such as
concentrated sugar solutions, which may also contain gum arabic,
talc, polyvinyl-pyrrolidone, carbopol gel, polyethylene glycol,
and/or titanium dioxide, lacquer solutions, and suitable organic
solvents or solvent mixtures. Dyestuffs or pigments may be added to
the tablets or dragee coatings for product identification or to
characterize the quantity of active compound, i.e., dosage.
[0115] Pharmaceutical preparations that can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a coating, such as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with filler
or binders, such as lactose or starches, lubricants, such as talc
or magnesium stearate, and, optionally, stabilizers. In soft
capsules, the active compounds may be dissolved or suspended in
suitable liquids, such as fatty oils, liquid, or liquid
polyethylene glycol with or without stabilizers.
[0116] Preferred sterile injectable preparations can be a solution
or suspension in a non-toxic parenterally acceptable solvent or
diluent. Examples of pharmaceutically acceptable carriers are
saline, buffered saline, isotonic saline (e.g. monosodium or
disodium phosphate, sodium, potassium; calcium or magnesium
chloride, or mixtures of such salts), Ringer's solution, dextrose,
water, sterile water, glycerol, ethanol, and combinations thereof
1,3-butanediol and sterile fixed oils are conveniently employed as
solvents or suspending media. Any bland fixed oil can be employed
including synthetic mono- or di-glycerides. Fatty acids such as
oleic acid also find use in the preparation of injectables.
[0117] The composition medium can also be a hydrogel, which is
prepared from any biocompatible or non-cytotoxic homo- or
hetero-polymer, such as a hydrophilic polyacrylic acid polymer that
can act as a drug absorbing sponge. Certain of them, such as, in
particular, those obtained from ethylene and/or propylene oxide are
commercially available. A hydrogel can be deposited directly onto
the surface of the tissue to be treated, for example during
surgical intervention.
[0118] Embodiments of pharmaceutical compositions of the present
invention comprise a replication defective recombinant viral vector
encoding the polynucleotide inhibitory agent of the present
invention and a transfection enhancer, such as poloxamer. An
example of a poloxamer is Poloxamer 407, which is commercially
available (BASF, Parsippany, N.J.) and is a non-toxic,
biocompatible polyol. A poloxamer impregnated with recombinant
viruses may be deposited directly on the surface of the tissue to
be treated, for example during a surgical intervention. Poloxamer
possesses essentially the same advantages as hydrogel while having
a lower viscosity.
[0119] The active expression-inhibiting agents may also be
entrapped in microcapsules prepared, for example, by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsules and poly-(methylmethacylate) microcapsules,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences (1980) 16th edition, Osol,
A. Ed.
[0120] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semi-permeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g. films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPO.TM.. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods. When encapsulated antibodies remain in
the body for a long time, they may denature or aggregate as a
result of exposure to moisture at 37.degree. C., resulting in a
loss of biological activity and possible changes in immunogenicity.
Rational strategies can be devised for stabilization depending on
the mechanism involved. For example, if the aggregation mechanism
is discovered to be intermolecular S--S bond formation through
thio-disulfide interchange, stabilization may be achieved by
modifying sulfhydryl residues, lyophilizing from acidic solutions,
controlling moisture content, using appropriate additives, and
developing specific polymer matrix compositions.
[0121] As defined above, therapeutically effective dose means that
amount of protein, polynucleotide, peptide, or its antibodies,
agonists or antagonists, which ameliorate the symptoms or
condition. Therapeutic efficacy and toxicity of such compounds can
be determined by standard pharmaceutical procedures in cell
cultures or experimental animals, e.g., ED50 (the dose
therapeutically effective in 50% of the population) and LD50 (the
dose lethal to 50% of the population). The dose ratio of toxic to
therapeutic effects is the therapeutic index, and it can be
expressed as the ratio, LD50/ED50. Pharmaceutical compositions that
exhibit large therapeutic indices are preferred. The data obtained
from cell culture assays and animal studies is used in formulating
a range of dosage for human use. The dosage of such compounds lies
preferably within a range of circulating concentrations that
include the ED50 with little or no toxicity. The dosage varies
within this range depending upon the dosage form employed,
sensitivity of the patient, and the route of administration.
[0122] For any compound, the therapeutically effective dose can be
estimated initially either in cell culture assays or in animal
models, usually mice, rabbits, dogs, or pigs. The animal model is
also used to achieve a desirable concentration range and route of
administration. Such information can then be used to determine
useful doses and routes for administration in humans. The exact
dosage is chosen by the individual physician in view of the patient
to be treated. Dosage and administration are adjusted to provide
sufficient levels of the active moiety or to maintain the desired
effect. Additional factors which may be taken into account include
the severity of the disease state, age, weight and gender of the
patient; diet, desired duration of treatment, method of
administration, time and frequency of administration, drug
combination(s), reaction sensitivities, and tolerance/response to
therapy. Long acting pharmaceutical compositions might be
administered every 3 to 4 days, every week, or once every two weeks
depending on half-life and clearance rate of the particular
formulation.
[0123] The pharmaceutical compositions according to this invention
may be administered to a subject by a variety of methods. They may
be added directly to tissues, complexed with cationic lipids,
packaged within liposomes, or delivered to eEF2 kinase-expressing
cells by other methods known in the art. Localized administration
to the desired tissues may be done by direct injection, transdermal
absorption, catheter, infusion pump or stent. The DNA, DNA/vehicle
complexes, or the recombinant virus particles are locally
administered to the site of treatment. Alternative routes of
delivery include, but are not limited to, intravenous injection,
intramuscular injection, subcutaneous injection, aerosol
inhalation, oral (tablet or pill form), topical, systemic, ocular,
intraperitoneal and/or intrathecal delivery. Examples of ribozyme
delivery and administration are provided in Sullivan et al. WO
94/02595.
[0124] Antibodies according to the invention may be delivered as a
bolus only, infused over time or both administered as a bolus and
infused over time. Those skilled in the art may employ different
formulations for polynucleotides than for proteins. Similarly,
delivery of polynucleotides or polypeptides will be specific to
particular cells, conditions, locations, etc.
[0125] As discussed hereinabove, recombinant viruses may be used to
introduce DNA encoding polynucleotide agents useful in the present
invention. Recombinant viruses according to the invention are
generally formulated and administered in the form of doses of
between about 10.sup.4 and about 10.sup.14 pfu. In the case of AAVs
and adenoviruses, doses of from about 10.sup.6 to about 10.sup.11
pfu are preferably used. The term pfu ("plaque-forming unit")
corresponds to the infective power of a suspension of virions and
is determined by infecting an appropriate cell culture and
measuring the number of plaques formed. The techniques for
determining the pfu titre of a viral solution are well documented
in the prior art.
[0126] The polypeptides or the polynucleotides employed in the
methods of the present invention may be free in solution, affixed
to a solid support, borne on a cell surface, or located
intracellularly. To perform the methods it is feasible to
immobilize either the polypeptide of the present invention or the
compound to facilitate separation of complexes from uncomplexed
forms of the polypeptide, as well as to accommodate automation of
the assay. Interaction (e.g., binding of) of the polypeptide of the
present invention with a compound can be accomplished in any vessel
suitable for containing the reactants. Examples of such vessels
include microtitre plates, test tubes, and microcentrifuge tubes.
In one embodiment, a fusion protein can be provided which adds a
domain that allows the polypeptide to be bound to a matrix. For
example, the polypeptide of the present invention can be "His"
tagged, and subsequently adsorbed onto Ni-NTA microtitre plates, or
ProtA fusions with the polypeptides of the present invention can be
adsorbed to IgG, which are then combined with the cell lysates
(e.g., (.sup.35S-labelled) and the candidate compound, and the
mixture incubated under conditions favorable for complex formation
(e.g., at physiological conditions for salt and pH). Following
incubation, the plates are washed to remove any unbound label, and
the matrix is immobilized. The amount of radioactivity can be
determined directly, or in the supernatant after dissociation of
the complexes. Alternatively, the complexes can be dissociated from
the matrix, separated by SDS-PAGE, and the level of the protein
binding to the protein of the present invention quantitated from
the gel using standard electrophoretic techniques.
[0127] Other techniques for immobilizing protein on matrices can
also be used in the method of identifying compounds. For example,
either the polypeptide of the present invention or the compound can
be immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated protein molecules of the present invention can be
prepared from biotin-NHS (N-hydroxy-succinimide) using techniques
well known in the art (e.g., biotinylation kit, Pierce Chemicals,
Rockford, Ill.), and immobilized in the wells of
streptavidin-coated 96 well plates (Pierce Chemical).
Alternatively, antibodies reactive with the polypeptides of the
present invention but which do not interfere with binding of the
polypeptide to the compound can be derivatized to the wells of the
plate, and the polypeptide of the present invention can be trapped
in the wells by antibody conjugation. As described above,
preparations of a labeled candidate compound are incubated in the
wells of the plate presenting the polypeptide of the present
invention, and the amount of complex trapped in the well can be
quantitated.
EXAMPLES
[0128] Using a mouse knockout model system, it has been determined
that loss of eEF2 kinase activity increases protein synthesis and
degradation rates and reduces damage to a cell or increases
resistance to damage to a cell. Mice lacking a functional eEF2
kinase appear and have normal development, behavior and
reproduction.
Example 1
Plasmids, Antibodies and Cells
[0129] Retroviral vector used for eEF2 kinase overexpression is
constructed by subcloning of eEF2 kinase cDNA from the pSIT
retroviral vector.sup.7 into LXSN vector (Clontech) using Eco
RI/XhoI cloning sites. Stable cell line is prepared through
infection of MEFs with pBabe-neo retroviral vector containing SV-40
large T antigen (a kind gift from Dr. J. Yuan). GSE 56 cell lines
are established using retroviral vector LXSP, containing
GSE56.sup.12. LXSP vector is prepared from LXSN vector by the
substitution of neomycin marker with puromycin. Antibodies against
p21 (F5) and p53 (Ab-1) are from Calbiochem Inc.; antibodies
against eEF2 and phospho-eEF2 are from Cell Signaling Inc. eEF2
kinase and eEF2 kinase.sup.+/+ primary mouse embryonic fibroblasts
used in this study are isolated from 10-12 day embryos following
standard protocols. Unless indicated, all cell lines are maintained
in DMEM with 10% fetal bovine serum.
Example 2
Transfection and Retroviral Infection
[0130] Packaging cells (Phoenix line) are plated in 60-mm plates
and transfected with 5 .mu.g of retroviral vector DNA using the
standard calcium phosphate procedure. Medium is changed after 8
hours. Virus-containing medium supplemented with 8 .mu.g of
Polybrene (Sigma) is collected at 24 and 48 hours post-transfection
and used for infection. Infected cells are selected for the
resistance to an appropriate selection agent.
Example 3
Western Analysis
[0131] For protein expression analysis, cells are washed twice with
ice-cold PBS, resuspended in lysis buffer (20 mM Na-phosphate [pH
7.5], 25 mM NaF, 1 mM orthovanadate, 5 mM EDTA), dissolved in
Laemmli SDS sample buffer and boiled for 10 minutes. Samples are
separated on 5-20% gradient SDS-PAGE and proteins are transferred
onto a PVDF membrane. Membranes are incubated with antibodies and
developed using ECL Plus reagents (Amersham Biosciences).
Example 4
Drugs and Reagents
[0132] Cells are exposed to different drugs and reagents:
ionomycin, hydrogen peroxide, doxorubicin and camptothecin (Sigma
Inc.) at indicated concentrations and time intervals. Low pH
effects are tested using DMEM medium buffered to pH 7, pH 6, pH 5
and pH 4 by 15 mM citric acid and 15 mM sodium phosphate. PFTa is
from Calbiochem Inc.
Example 5
Drug Sensitivity Assays
[0133] MTT Survival Assay: MEFs used in this study
(2.times.10.sup.3 cells per well of a 96 well plate) are incubated
in the presence of indicated drugs for 24 hours. Cell viability is
determined using the standard MTT assay.sup.30. Each experiment is
repeated three times for each drug and each cell line using three
parallel wells for each drug concentration. For colony formation
assay cells are treated with indicated drugs for 24 hours and
replated with complete DMEM at low density (500 cells per well in
6-well plate) in duplicate. After 10 days colonies are stained with
10 mg/ml methylene blue (Sigma Inc.) in 50% methanol.
Example 6
DNA Ladders, Annexin V Staining and Flow Cytometry
[0134] DNA fragmentation pattern (DNA laddering): DNA is analyzed
by agarose gel electrophoresis. Cells are incubated in a serum-free
media for 48 hours, scraped and centrifuged at 1200 rpm for 10 min.
The cell pellets are resuspended in 1 ml of lysis buffer (10 mM
Tris-HCl, pH 7.4, 10 mM NaCl, 10 mM EDTA, 100 .mu.g/ml proteinase
K) and incubated for 2 hours at 50.degree. C. DNA is extracted with
equal volume of phenol-chloroform-isoamyl alcohol. The aqueous
phase is precipitated with 2.5 volumes of ice-cold ethanol and 10%
volume of 3 M sodium acetate, pH5.2 at -20.degree. C. overnight and
analyzed by agarose gel electrophoresis. Flow cytometry performed
using annexin V staining: cells are incubated with 600 ng/ml of
doxorubicin for 24 hours, then harvested by trypsin digestion,
washed with PBS and stained for annexin V assay according to
manufacturer's instructions (BD Biosciences, CA). The fluorescence
is analyzed by flow cytometry instrument (FASC Scan Cytomics FC500,
Beckman Coulter Inc.) and CXP software.
Example 7
eEF2 Kinase Knockout Mouse
[0135] In order to investigate the function of eEF2 kinase an eEF2
kinase knockout mouse is generated by disrupting the eEF2 kinase
gene in mouse embryonic stem (ES) cells. A targeting vector is
used, in which exon 7 and the majority of exon 8 are replaced with
the neomycin resistance gene (FIG. 1a), resulting in the
elimination of a portion of the catalytic domain of eEF2 kinase
(FIG. 1a, b; FIG. 2A, 2B). eEF2 kinase.sup.+/- ES cell clones are
identified and used to obtain chimeras and subsequently
eEF2k.sup.+/- mice. Mating of eEF2 kinase.sup.+/- mice produced
progeny of eEF2 kinase.sup.+/-, eEF2 kinase.sup.+/+ and eEF2
kinase.sup.-/- mice with the expected ratio of 2:1:1. No
phosphorylated eEF2 is observed in the tissue extracts of the eEF2
kinase.sup.-/- mice, indicating their complete lack of eEF2 kinase
activity (FIG. 1c). The knockout mice are viable, do not have
visible abnormalities and give normal progeny for many
generations.
[0136] eEF2 kinase is a Ca.sup.2.+-./calmodulin-dependent enzyme
whose cellular activity is previously demonstrated to increase upon
incubation with Ca.sup.2+ ionophores ionomycin or A23187, which can
induce apoptotic cell death. The possible correlation between
activation of eEF2 kinase and the resultant phosphorylation of eEF2
and Ca.sup.2+ ionophore-induced cell death is addressed by
comparing the viability of wild type and eEF2 kinase knockout mouse
embryonic fibroblasts (MEFs) after incubation of each with
different concentrations of ionomycin. As shown in FIG. 1d,
exposure of eEF2 kinase.sup.+/+ MEFs to increasing concentrations
of ionomycin from 1 to 10 .mu.M resulted in a progressive decrease
of cell viability. Notably, approximately 70% of cells die after
incubation for 24 h with 10 .mu.M ionomycin, whereas the same
concentrations of ionomycin have virtually no effect on the
viability of eEF2 kinase.sup.-/- MEFs. These results suggest that
activation of eEF2 kinase may facilitate cell death induced by
ionomycin and that inactivation of eEF2 kinase results in an
increased resistance to damage caused by Ca.sup.2+ ionophores.
[0137] eEF2 kinase can also be activated by acidic pH. Acidic pH is
cytotoxic and therefore activation of eEF2 kinase by acidic pH can
be involved in the regulation of cell death. The effect of acidic
pH on the viability of eEF2 kinase knockout and wild types MEFs is
also analyzed. FIG. 1e shows that eEF2 kinase.sup.-/- MEFs are
significantly more resistant than wild type MEFs to the cytotoxic
effect of acidic pH. The increased viability of eEF2 kinase
knockout cells in an acidic environment is also observed in the
stable cell lines derived from MEFs. Using a low-density clonogenic
assay it is shown that after incubation at pH 6 for 3 hours, eEF2
kinase knockout cells produce significantly more colonies than wild
type cells (FIG. 1g). eEF2 kinase.sup.-/- MEFs are also
significantly more resistant than wild type MEFs to hydrogen
peroxide (FIG. 1f). Thus, these results demonstrate that knockout
of eEF2 kinase increases resistance of cells to stress, induced by
Ca.sup.2+ ionophore and acidic pH, as well as to oxidative stress
induced by hydrogen peroxide.
Example 8
Sensitivity to Chemotherapeutic Drugs
[0138] Next the sensitivity of knockout and wild type MEFs to the
chemotherapeutic drugs camptothecin (CPT) and doxorubicin (DOX) is
analyzed. As shown in FIGS. 3a and 3b, eEF2 kinase.sup.-/- MEFs and
their stable cell lines are significantly more resistant than their
wild type counterparts to CPT and DOX, respectively. Introduction
of eEF2 kinase cDNA into eEF2 kinase knockout cells restored
sensitivity of these cells to DOX to the level observed in wild
type cells (FIG. 3c).
[0139] To investigate the mode of cell death that is affected by
eEF2 an annexin V assay and DNA fragmentation analysis is
performed. As shown in FIG. 3d the percentage of apoptotic annexin
V positive cells after 24 hours of treatment with doxorubicin is
significantly lower in eEF2 kinase cells than in eEF2
kinase.sup.+/+ cells. Apoptotic DNA ladder formation in wild type
and eEF2 kinase knockout cells in response to serum starvation is
also analyzed. Incubation of eEF2 kinase.sup.+/+ cells in serum
free media for 48 h resulted in significant DNA fragmentation,
whereas no DNA ladder formation is observed in eEF2 kinase.sup.-/-
cells incubated under the same conditions (FIG. 3e). This suggests
that the absence of eEF2 kinase results in inhibition of apoptosis
and therefore eEF2 kinase might be a factor that facilitates
apoptosis.
[0140] Since the tumor suppressor p53 is known to be involved in
modulating the sensitivity of MEFs to various cytotoxic drugs and
induction of apoptosis, it is determined whether the effects of the
eEF2 kinase knockout on cell sensitivity to doxorubicin might
depend on functional p53. Sensitivity to DOX is assessed in wild
type and eEF2 kinase knockout MEFs in which p53 is inactivated by
overexpression of GSE 56, a carboxyl-terminal portion of p53 that
acts as a dominant negative p53 mutant, or by incubation with
pifithrin .alpha. (PFT .alpha.), a chemical p53 inhibitor.
Inactivation of p53 either by GSE56 or PFT.alpha. only slightly
affected drug sensitivity of eEF2 kinase knockout fibroblasts,
while strongly decreasing sensitivity of wild type cells (FIG. 4a,
b), suggesting that the effect of eEF2 kinase on drug sensitivity
depends on functional p53. Additionally, the effect of DOX
treatment on the induction of p53 and cyclin-dependent kinase
inhibitor p21 (WAF1), whose expression is known to be regulated by
p53, is determined. The expression of p53 is undetectable in both
untreated eEF2 kinase.sup.-/- and untreated wild type MEFs. After
treatment with 600 ng/ml of DOX for 24 h, p53 is similarly induced
in both eEF2 kinase.sup.-/- and eEF2 kinase.sup.+/+ MEFs. However,
p21 WAF1 is induced significantly more strongly in eEF2
kinase.sup.+/+ MEFs (FIGS. 3c and 3d). Expression of several p53
dependent genes in eEF2 kinase knockout and wild type cells is also
determined after treatment with doxorubicin using RT-PCR. In
addition to p21 WAF1, the induction of apoptosis-related genes,
GADD45 and PIG3 is significantly higher in eEF2 kinase.sup.+/+ DOX
treated cells than in eEF2 kinase.sup.-/- DOX treated cells. These
results suggest that although induction of p53 in eEF2 kinase
knockout cells is comparable to that of the wild type, its
transactivation activity is altered.
[0141] Long-term survival assays in eEF2 kinase.sup.+/-, eEF2
kinase.sup.+/+ and eEF2 kinase.sup.-/- mice reveal that knockout of
eEF2 kinase results in a significant increase in maximal life span
(FIG. 5a, b). Maximal lifespan, defined as the average age of the
last 10% of surviving mice, is increased by approximately 30% in
eEF2 kinase.sup.-/- mice (36.6 month) and approximately 18% in eEF2
kinase.sup.+/- mice (33.1 month) in comparison with eEF2
kinase.sup.+/+ mice (28 month). Since increase in maximal life span
is observed in both eEF2 kinase.sup.-/- and eEF2 kinase.sup.+/-
mice, the complete elimination of eEF2 kinase is not required for
the life span extending effect. The significant increase in maximal
life span in eEF2 kinase knockout mice is not accompanied by an
increase in median life span. Maximal life span is considered to be
a key parameter in the measurement of longevity and its extension
indicates a genuine slowing of the aging process. In contrast to
maximal life span, which depends on the cumulative effect of many
different factors related to aging, median life span is often
determined by a single factor, that causes death in the majority of
animals in the population and which may or may not be related to
aging. Therefore the increase in maximal, but not median life span
in eEF2 kinase.sup.-/- and eEF2 kinase.sup.+/- mice suggests that
the decrease of eEF2 kinase affects aging per se.
[0142] The increased maximal life span in eEF2 kinase knockout mice
can be related to increased cellular stress resistance. In addition
there is a correlation between life span of various mammalian
species and stress resistance of fibroblasts derived from them.
[0143] Increased maximal life span in eEF2 kinase knockout mice can
also be related to altered regulation of p53 and p21. Increased
activation of p53 is known to cause premature aging in mice and
overexpression of p21 results in the induction of various genes
associated with senescence and aging, including p66.sup.SHC.
Therefore, the reduced activation of p53 observed in eEF2
kinase.sup.-/- cells and the reduced induction of p21 that it leads
to, can contribute to increased longevity in eEF2 kinase knockout
mice.
Example 9
Sensitivity to Radiation
[0144] Gamma irradiation of mice: 8 to 12 week old mice are
irradiated at a dose of 8 grays (Gy) of whole-body gamma
irradiation produced by Caesium-137 source (Nordiom gammacell 40).
Each cohort of mice consists of 10 mice including 5 males and 5
females.
[0145] Preparation of MEFs: Mouse embryonic fibroblasts (MEFs) are
prepared from E13.5 embryos and immortalized by large T antigen via
retrovirus infection. Virus is collected from the medium of
transient triple-transfected 239T cells by three plasmids including
VSV, gal/pol, and pBebe-neo TcDNA.
[0146] TUNEL assay: Cells are treated with or without 1.6 .mu.M of
doxorubicin for 24 hours. After treatment, cells are collected and
fixed in 1% paraformaldehyde for 15 minutes on ice. Cells are
stored in 75% ethanol at -20.degree. C. until staining, which is
performed according to the manufacturer's instructions (In Situ
Cell Death Detection kit, Roche). Apoptotic cells are labeled with
fluorescein and counted by flow cytometry.
[0147] The effect of eEF2 kinase deficiency on the short-term
survival of mice under stress is analyzed. Mice are irradiated with
8 Gy of whole-body .gamma.-irradiation. After irradiation, 50% of
wild type mice die within 16 days; however none of the
eEF2K-deficient mice die during the same interval (FIG. 6a). Within
one month after irradiation the hair color of surviving, normally
black wild type mice turns grey but, unexpectedly, this does not
occur in eEF2 kinase deficient mice (FIG. 6b). In addition,
significant hair loss is noted in irradiated wild type mice, but
not in irradiated eEF2K-/- mice.
[0148] Because .gamma.-irradiation is known to induce apoptosis,
the increased resistance to .gamma.-irradiation observed in
eEF2K-deficient mice may be due to a corresponding increase in the
resistance of eEF2K-deficient cells to apoptosis. To test this
possibility, the effect of eEF2K deficiency on apoptosis in cells
isolated from eEF2K-/- mice is analyzed. Cells from eEF2K deficient
mice are significantly more resistant to apoptosis induced by
doxorubicin or hydrogen peroxide. As can be seen in FIG. 7a,
significant cell death is observed in wild type mouse embryonic
fibroblasts (MEFs) treated for 24 hours with 1.6 .mu.M doxorubicin.
However, much less cell death is observed in eEF2K-deficient cells
treated in the same manner. The results of the TUNEL assay suggest
that the reduction in cell death in eEF2K-deficient cells is due to
decreased apoptosis (FIG. 7b). To verify that the decreased
apoptosis is due to the absence of eEF2K, eEF2K cDNA is introduced
into eEF2K-deficient MEFs. As can be seen in FIG. 7c, after
treatment with hydrogen peroxide or doxorubicin, MEFs carrying
eEF2K cDNA have more activated caspase 3 than eEF2K-deficient cells
from which they are derived, thus confirming that eEF2K enhances
apoptosis.
Example 10
Distribution of Phosphorylated eEF2
[0149] Western blot and immunohistochemistry: Antibodies against
phosphorylated eEF2 and cleaved caspase 3 (5A1) are purchased from
Cell SignalingTech. Antibody against mouse eEF2K is purchased from
BD Biosciences. Antibody against actin (AC-40) is purchased from
Sigma. Mouse liver tissue is obtained from eEF2K knock-out and
aged-matched wild type adult mice and lysed in buffer with 50 mM
Tris-HCl in pH 8.0, 2 mM EDTA, 75 mM NaCl, 0.05 mM DTT, 1 mM PMSF,
0.5% Triton X-100, 10% glycerol, and 1 tablet of protease inhibitor
cocktail (CPI, Roche)/10 ml. After H.sub.2O.sub.2 or doxorubicin
treatment, cells are lysed in SDS containing buffer (20 mM HEPES in
pH 7.5, 50 mM NaCl, mM KCl, 10 mM DTT, 3 mM benzamidine, 1% SDS, 1
mM sodium orthovanadate, sodium pyrophosphate, 1 tablet of CPI/10
ml). Western blotting and immunohistochemistry are performed
according to the manufacturer's instructions (Cell
SignalingTech).
[0150] Using antibody that specifically recognizes phosphorylated
eEF2, the distribution of phosphorylated eEF2 in tissue culture
cells and in various human tissues is analyzed. Western blot
analysis reveals strong and persistent phosphorylation of eEF2 in
NIH3T3 cells treated with hydrogen peroxide for various time
periods (FIG. 8a). Immunocytochemical analysis of these cells show
that levels of phosphorylated eEF2 are high in rounding cells and
highest in cells undergoing apoptosis, whereas no significant
phosphorylation of eEF2 is detected in control cells grown under
standard conditions (FIG. 8b). Similarly, treatment of HeLa cells
with hydrogen peroxide results in a dramatic and selective increase
in phosphorylation of eEF2 in apoptotic cells with condensed
chromatin (FIG. 8c).
[0151] Various human tissues are also examined for the presence of
phosphorylated eEF2 using human multiple tissue arrays.
Phosphorylated eEF2 is not detectable in most tissues. However,
significant phosphorylation of eEF2 in lymph nodes is observed
(FIGS. 8d-e). Particularly intense phosphorylation is found in
macrophages that likely represent staining of phagocytized
lymphocytes undergoing apoptosis (FIG. 8e). Phosphorylated eEF2 is
also detected in pyramidal neurons in histological sections of
Alzheimer's disease brains (FIG. 80, but not in neurons of
neurologically normal, age-matched control brains (FIG. 8g). These
results suggest that activation of eEF2 kinase is associated with
the cellular response to stress and cell death.
[0152] The foregoing examples and description of the preferred
embodiments should be taken as illustrating, rather than as
limiting the present invention as defined by the claims. As will be
readily appreciated, numerous variations and combinations of the
features set forth above can be utilized without departing from the
present invention as set forth in the claims. Such variations are
not regarded as a departure from the spirit and script of the
invention, and all such variations are intended to be included
within the scope of the following claims.
Sequence CWU 1
1
412178DNAHomo sapiens 1atggcagacg aagacctcat cttccgcctg gaaggtgttg
atggcggcca gtccccccga 60gctggccatg atggtgattc tgatggggac agcgacgatg
aggaaggtta cttcatctgc 120cccatcacgg atgacccaag ctcgaaccag
aatgtcaatt ccaaggttaa taagtactac 180agcaacctaa caaaaagtga
gcggtatagc tccagcgggt ccccggcaaa ctccttccac 240ttcaaggaag
cctggaagca cgcaatccag aaggccaagc acatgcccga cccctgggct
300gagttccacc tggaagatat tgccaccgaa cgtgctactc gacacaggta
caacgccgtc 360accggggaat ggctggatga tgaagttctg atcaagatgg
catctcagcc cttcggccga 420ggagcaatga gggagtgctt ccggacgaag
aagctctcca acttcttgca tgcccagcag 480tggaagggcg cctccaacta
cgtggcgaag cgctacatcg agcccgtaga ccgggatgtg 540tactttgagg
acgtgcgtct acagatggag gccaagctct ggggggagga gtataatcgg
600cacaagcccc ccaagcaggt ggacatcatg cagatgtgca tcatcgagct
gaaggacaga 660ccgggcaagc ccctcttcca cctggagcac tacatcgagg
gcaagtacat caagtacaac 720tccaactctg gctttgtccg tgatgacaac
atccgactga cgccgcaggc cttcagccac 780ttcacttttg agcgttccgg
ccatcagctg atagtggtgg acatccaggg agttggggat 840ctctacactg
acccacagat ccacacggag acgggcactg actttggaga cggcaaccta
900ggtgtccgcg ggatggcgct cttcttctac tctcatgcct gcaaccggat
ttgcgagagc 960atgggccttg ctccctttga cctctcgccc cgggagaggg
atgcagtgaa tcagaacacc 1020aagctgctgc aatcagccaa gaccatcttg
agaggaacag aggaaaaatg tgggagcccc 1080cgagtaagga ccctctctgg
gagccggcca cccctgctcc gtcccctttc agagaactct 1140ggagacgaga
acatgagcga cgtgaccttc gactctctcc cttcttcccc atcttcggcc
1200acaccacaca gccagaagct agaccacctc cattggccag tgttcagtga
cctcgataac 1260atggcatcca gagaccatga tcatctagac aaccaccggg
agtctgagaa tagtggggac 1320agcggatacc ccagtgagaa gcggggtgag
ctggatgacc ctgagccccg agaacatggc 1380cactcataca gtaatcggaa
gtacgagtct gacgaagaca gcctgggcag ctctggacgg 1440gtatgtgtag
agaagtggaa tctcctcaac tcctcccgcc tccacctgcc gagggcttcg
1500gccgtggccc tggaagtgca aaggcttaat gctctggacc tcgaaaagaa
aatcgggaag 1560tccattttgg ggaaggtcca tctggccatg gtgcgctacc
acgagggtgg gcgcttctgc 1620gagaagggcg aggagtggga ccaggagtcg
gctgtcttcc acctggagca cgcagccaac 1680ctgggcgagc tggaggccat
cgtgggcctg ggactcatgt actcgcagtt gcctcatcac 1740atcctagccg
atgtctctct gaaggagaca gaagagaaca aaaccaaagg atttgattac
1800ttactaaagg ccgctgaagc tggcgacagg cagtccatga tcctagtggc
gcgagctttt 1860gactctggcc agaacctcag cccggacagg tgccaagact
ggctagaggc cctgcactgg 1920tacaacactg ccctggagat gacggactgt
gatgagggcg gtgagtacga cggaatgcag 1980gacgagcccc ggtacatgat
gctggccagg gaggcagaga tgctgttcac aggaggctac 2040gggctggaga
aggacccgca gagatcaggg gacttgtata cccaggcagc agaggcagcg
2100atggaagcca tgaagggccg actggccaac cagtactacc aaaaggctga
agaggcctgg 2160gcccagatgg aggaataa 21782725PRTHomo sapiens 2Met Ala
Asp Glu Asp Leu Ile Phe Arg Leu Glu Gly Val Asp Gly Gly 1 5 10 15
Gln Ser Pro Arg Ala Gly His Asp Gly Asp Ser Asp Gly Asp Ser Asp 20
25 30 Asp Glu Glu Gly Tyr Phe Ile Cys Pro Ile Thr Asp Asp Pro Ser
Ser 35 40 45 Asn Gln Asn Val Asn Ser Lys Val Asn Lys Tyr Tyr Ser
Asn Leu Thr 50 55 60 Lys Ser Glu Arg Tyr Ser Ser Ser Gly Ser Pro
Ala Asn Ser Phe His 65 70 75 80 Phe Lys Glu Ala Trp Lys His Ala Ile
Gln Lys Ala Lys His Met Pro 85 90 95 Asp Pro Trp Ala Glu Phe His
Leu Glu Asp Ile Ala Thr Glu Arg Ala 100 105 110 Thr Arg His Arg Tyr
Asn Ala Val Thr Gly Glu Trp Leu Asp Asp Glu 115 120 125 Val Leu Ile
Lys Met Ala Ser Gln Pro Phe Gly Arg Gly Ala Met Arg 130 135 140 Glu
Cys Phe Arg Thr Lys Lys Leu Ser Asn Phe Leu His Ala Gln Gln 145 150
155 160 Trp Lys Gly Ala Ser Asn Tyr Val Ala Lys Arg Tyr Ile Glu Pro
Val 165 170 175 Asp Arg Asp Val Tyr Phe Glu Asp Val Arg Leu Gln Met
Glu Ala Lys 180 185 190 Leu Trp Gly Glu Glu Tyr Asn Arg His Lys Pro
Pro Lys Gln Val Asp 195 200 205 Ile Met Gln Met Cys Ile Ile Glu Leu
Lys Asp Arg Pro Gly Lys Pro 210 215 220 Leu Phe His Leu Glu His Tyr
Ile Glu Gly Lys Tyr Ile Lys Tyr Asn 225 230 235 240 Ser Asn Ser Gly
Phe Val Arg Asp Asp Asn Ile Arg Leu Thr Pro Gln 245 250 255 Ala Phe
Ser His Phe Thr Phe Glu Arg Ser Gly His Gln Leu Ile Val 260 265 270
Val Asp Ile Gln Gly Val Gly Asp Leu Tyr Thr Asp Pro Gln Ile His 275
280 285 Thr Glu Thr Gly Thr Asp Phe Gly Asp Gly Asn Leu Gly Val Arg
Gly 290 295 300 Met Ala Leu Phe Phe Tyr Ser His Ala Cys Asn Arg Ile
Cys Glu Ser 305 310 315 320 Met Gly Leu Ala Pro Phe Asp Leu Ser Pro
Arg Glu Arg Asp Ala Val 325 330 335 Asn Gln Asn Thr Lys Leu Leu Gln
Ser Ala Lys Thr Ile Leu Arg Gly 340 345 350 Thr Glu Glu Lys Cys Gly
Ser Pro Arg Val Arg Thr Leu Ser Gly Ser 355 360 365 Arg Pro Pro Leu
Leu Arg Pro Leu Ser Glu Asn Ser Gly Asp Glu Asn 370 375 380 Met Ser
Asp Val Thr Phe Asp Ser Leu Pro Ser Ser Pro Ser Ser Ala 385 390 395
400 Thr Pro His Ser Gln Lys Leu Asp His Leu His Trp Pro Val Phe Ser
405 410 415 Asp Leu Asp Asn Met Ala Ser Arg Asp His Asp His Leu Asp
Asn His 420 425 430 Arg Glu Ser Glu Asn Ser Gly Asp Ser Gly Tyr Pro
Ser Glu Lys Arg 435 440 445 Gly Glu Leu Asp Asp Pro Glu Pro Arg Glu
His Gly His Ser Tyr Ser 450 455 460 Asn Arg Lys Tyr Glu Ser Asp Glu
Asp Ser Leu Gly Ser Ser Gly Arg 465 470 475 480 Val Cys Val Glu Lys
Trp Asn Leu Leu Asn Ser Ser Arg Leu His Leu 485 490 495 Pro Arg Ala
Ser Ala Val Ala Leu Glu Val Gln Arg Leu Asn Ala Leu 500 505 510 Asp
Leu Glu Lys Lys Ile Gly Lys Ser Ile Leu Gly Lys Val His Leu 515 520
525 Ala Met Val Arg Tyr His Glu Gly Gly Arg Phe Cys Glu Lys Gly Glu
530 535 540 Glu Trp Asp Gln Glu Ser Ala Val Phe His Leu Glu His Ala
Ala Asn 545 550 555 560 Leu Gly Glu Leu Glu Ala Ile Val Gly Leu Gly
Leu Met Tyr Ser Gln 565 570 575 Leu Pro His His Ile Leu Ala Asp Val
Ser Leu Lys Glu Thr Glu Glu 580 585 590 Asn Lys Thr Lys Gly Phe Asp
Tyr Leu Leu Lys Ala Ala Glu Ala Gly 595 600 605 Asp Arg Gln Ser Met
Ile Leu Val Ala Arg Ala Phe Asp Ser Gly Gln 610 615 620 Asn Leu Ser
Pro Asp Arg Cys Gln Asp Trp Leu Glu Ala Leu His Trp 625 630 635 640
Tyr Asn Thr Ala Leu Glu Met Thr Asp Cys Asp Glu Gly Gly Glu Tyr 645
650 655 Asp Gly Met Gln Asp Glu Pro Arg Tyr Met Met Leu Ala Arg Glu
Ala 660 665 670 Glu Met Leu Phe Thr Gly Gly Tyr Gly Leu Glu Lys Asp
Pro Gln Arg 675 680 685 Ser Gly Asp Leu Tyr Thr Gln Ala Ala Glu Ala
Ala Met Glu Ala Met 690 695 700 Lys Gly Arg Leu Ala Asn Gln Tyr Tyr
Gln Lys Ala Glu Glu Ala Trp 705 710 715 720 Ala Gln Met Glu Glu 725
32175DNAMus musculus 3atggcagacg aagacctcat cttctgcctg gaaggtgttg
acggtggcag gtgctcccga 60gctggccaca atgcggactc tgacacagac agtgacgatg
atgagggcta tttcatctgc 120cccatcactg atgaccacat gtccaatcag
aatgtcagct ccaaagtcca gagctactat 180agcaacctaa caaaaacaga
gtgcggctcc acagggtcac cagccagctc cttccacttc 240aaggaagcct
ggaagcatgc gatcgagaaa gccaagcaca tgcctgaccc ctgggctgaa
300ttccatctcg aggacatcgc cacagaacat gctactcggc acaggtacaa
cgctgtcacc 360ggggaatggc tgaaagacga ggttctgatc aagatggcgt
ctcagccctt cggccgtgga 420gcaatgaggg agtgcttcag gacgaagaaa
ctctccaact tcttgcacgc ccagcaatgg 480aagggggcct ccaactacgt
ggccaagcgc tacatcgagc cggtggacag gagcgtgtac 540tttgaggatg
tgcagctcca gatggaggcg aagctctggg gggaggatta caatcggcac
600aagcccccca agcaggtgga tatcatgcag atgtgcatca ttgagctaaa
ggacagacca 660ggccagcccc tcttccactt ggagcactac attgagggca
agtacatcaa gtacaattcc 720aactcaggct ttgtccgtga tgacaacatc
cgactaaccc cacaggcctt cagccatttc 780acatttgagc gttctggtca
tcagctgatt gtagtggaca tccagggtgt gggtgacctt 840tataccgacc
cacagatcca cactgagaaa ggcactgact ttggagatgg taaccttggt
900gtccggggaa tggctctctt cttctactct catgcctgca accggatttg
tcagagcatg 960ggccttacgc cctttgacct ctccccacgg gaacaggatg
cggtgaatca gagcaccagg 1020ctattgcaat cagccaagac catcttgagg
gggacagagg agaagtgtgg gagtccccgc 1080ataaggacac tctctagcag
ccggccccct ttgctccttc gcctgtcaga gaactccggg 1140gatgagaaca
tgagtgacgt gacctttgac tctctgcctt cctccccgtc ttcagctaca
1200ccacacagcc agaaactgga ccacctccat tggccagtgt ttggtgacct
cgataacatg 1260ggccctagag accatgaccg tatggacaat caccgggact
ctgagaatag tggggacagt 1320gggtatccaa gcgagaagcg aagtgacctg
gatgatcctg agccccgaga acacggccac 1380tccaacggca accgaaggca
tgaatctgac gaggatagcc tgggcagctc tggacgggtc 1440tgtgtggaga
cgtggaacct gctcaatccc tcccgcctgc acctgccgag gccctcggcc
1500gtggccctag aagtgcagag gctaaatgcc ctggaccttg gaaggaaaat
cgggaagtct 1560gttttgggga aagtccattt ggccatggtg cgataccacg
agggcgggcg cttctgcgag 1620aaggatgagg agtgggatcg agagtcagcc
atcttccatc tggagcatgc agctgacctg 1680ggagaactgg aggccatcgt
gggcctaggc ctcatgtact ctcagctgcc ccaccacatc 1740ctggctgatg
tctctctgaa ggagacagag gagaacaaga caaaaggctt tgattactta
1800ctgaaggcgg cagaagctgg tgacaggcat tccatgattt tagtggcccg
agcttttgac 1860actggcctga acctcagccc agacaggtgt caagactggt
cggaagcctt gcactggtac 1920aacacagccc tggagacaac agactgcgat
gaaggcgggg agtacgatgg gatacaggac 1980gagccccagt acgcactgct
ggccagggag gcggagatgc tgctcaccgg gggatttgga 2040ctggacaaga
acccccaaag atcaggagat ttgtacaccc aggcagctga ggcagcaatg
2100gaagccatga agggccggct agccaaccag tactacgaga aggcggaaga
ggcctgggcc 2160cagatggagg aataa 21754724PRTMus musculus 4Met Ala
Asp Glu Asp Leu Ile Phe Cys Leu Glu Gly Val Asp Gly Gly 1 5 10 15
Arg Cys Ser Arg Ala Gly His Asn Ala Asp Ser Asp Thr Asp Ser Asp 20
25 30 Asp Asp Glu Gly Tyr Phe Ile Cys Pro Ile Thr Asp Asp His Met
Ser 35 40 45 Asn Gln Asn Val Ser Ser Lys Val Gln Ser Tyr Tyr Ser
Asn Leu Thr 50 55 60 Lys Thr Glu Cys Gly Ser Thr Gly Ser Pro Ala
Ser Ser Phe His Phe 65 70 75 80 Lys Glu Ala Trp Lys His Ala Ile Glu
Lys Ala Lys His Met Pro Asp 85 90 95 Pro Trp Ala Glu Phe His Leu
Glu Asp Ile Ala Thr Glu His Ala Thr 100 105 110 Arg His Arg Tyr Asn
Ala Val Thr Gly Glu Trp Leu Lys Asp Glu Val 115 120 125 Leu Ile Lys
Met Ala Ser Gln Pro Phe Gly Arg Gly Ala Met Arg Glu 130 135 140 Cys
Phe Arg Thr Lys Lys Leu Ser Asn Phe Leu His Ala Gln Gln Trp 145 150
155 160 Lys Gly Ala Ser Asn Tyr Val Ala Lys Arg Tyr Ile Glu Pro Val
Asp 165 170 175 Arg Ser Val Tyr Phe Glu Asp Val Gln Leu Gln Met Glu
Ala Lys Leu 180 185 190 Trp Gly Glu Asp Tyr Asn Arg His Lys Pro Pro
Lys Gln Val Asp Ile 195 200 205 Met Gln Met Cys Ile Ile Glu Leu Lys
Asp Arg Pro Gly Gln Pro Leu 210 215 220 Phe His Leu Glu His Tyr Ile
Glu Gly Lys Tyr Ile Lys Tyr Asn Ser 225 230 235 240 Asn Ser Gly Phe
Val Arg Asp Asp Asn Ile Arg Leu Thr Pro Gln Ala 245 250 255 Phe Ser
His Phe Thr Phe Glu Arg Ser Gly His Gln Leu Ile Val Val 260 265 270
Asp Ile Gln Gly Val Gly Asp Leu Tyr Thr Asp Pro Gln Ile His Thr 275
280 285 Glu Lys Gly Thr Asp Phe Gly Asp Gly Asn Leu Gly Val Arg Gly
Met 290 295 300 Ala Leu Phe Phe Tyr Ser His Ala Cys Asn Arg Ile Cys
Gln Ser Met 305 310 315 320 Gly Leu Thr Pro Phe Asp Leu Ser Pro Arg
Glu Gln Asp Ala Val Asn 325 330 335 Gln Ser Thr Arg Leu Leu Gln Ser
Ala Lys Thr Ile Leu Arg Gly Thr 340 345 350 Glu Glu Lys Cys Gly Ser
Pro Arg Ile Arg Thr Leu Ser Ser Ser Arg 355 360 365 Pro Pro Leu Leu
Leu Arg Leu Ser Glu Asn Ser Gly Asp Glu Asn Met 370 375 380 Ser Asp
Val Thr Phe Asp Ser Leu Pro Ser Ser Pro Ser Ser Ala Thr 385 390 395
400 Pro His Ser Gln Lys Leu Asp His Leu His Trp Pro Val Phe Gly Asp
405 410 415 Leu Asp Asn Met Gly Pro Arg Asp His Asp Arg Met Asp Asn
His Arg 420 425 430 Asp Ser Glu Asn Ser Gly Asp Ser Gly Tyr Pro Ser
Glu Lys Arg Ser 435 440 445 Asp Leu Asp Asp Pro Glu Pro Arg Glu His
Gly His Ser Asn Gly Asn 450 455 460 Arg Arg His Glu Ser Asp Glu Asp
Ser Leu Gly Ser Ser Gly Arg Val 465 470 475 480 Cys Val Glu Thr Trp
Asn Leu Leu Asn Pro Ser Arg Leu His Leu Pro 485 490 495 Arg Pro Ser
Ala Val Ala Leu Glu Val Gln Arg Leu Asn Ala Leu Asp 500 505 510 Leu
Gly Arg Lys Ile Gly Lys Ser Val Leu Gly Lys Val His Leu Ala 515 520
525 Met Val Arg Tyr His Glu Gly Gly Arg Phe Cys Glu Lys Asp Glu Glu
530 535 540 Trp Asp Arg Glu Ser Ala Ile Phe His Leu Glu His Ala Ala
Asp Leu 545 550 555 560 Gly Glu Leu Glu Ala Ile Val Gly Leu Gly Leu
Met Tyr Ser Gln Leu 565 570 575 Pro His His Ile Leu Ala Asp Val Ser
Leu Lys Glu Thr Glu Glu Asn 580 585 590 Lys Thr Lys Gly Phe Asp Tyr
Leu Leu Lys Ala Ala Glu Ala Gly Asp 595 600 605 Arg His Ser Met Ile
Leu Val Ala Arg Ala Phe Asp Thr Gly Leu Asn 610 615 620 Leu Ser Pro
Asp Arg Cys Gln Asp Trp Ser Glu Ala Leu His Trp Tyr 625 630 635 640
Asn Thr Ala Leu Glu Thr Thr Asp Cys Asp Glu Gly Gly Glu Tyr Asp 645
650 655 Gly Ile Gln Asp Glu Pro Gln Tyr Ala Leu Leu Ala Arg Glu Ala
Glu 660 665 670 Met Leu Leu Thr Gly Gly Phe Gly Leu Asp Lys Asn Pro
Gln Arg Ser 675 680 685 Gly Asp Leu Tyr Thr Gln Ala Ala Glu Ala Ala
Met Glu Ala Met Lys 690 695 700 Gly Arg Leu Ala Asn Gln Tyr Tyr Glu
Lys Ala Glu Glu Ala Trp Ala 705 710 715 720 Gln Met Glu Glu
* * * * *