U.S. patent application number 10/457614 was filed with the patent office on 2004-04-29 for cell-permeable peptide inhibitors of the jnk signal transduction pathway.
Invention is credited to Bonny, Christophe.
Application Number | 20040082509 10/457614 |
Document ID | / |
Family ID | 46299396 |
Filed Date | 2004-04-29 |
United States Patent
Application |
20040082509 |
Kind Code |
A1 |
Bonny, Christophe |
April 29, 2004 |
Cell-permeable peptide inhibitors of the JNK signal transduction
pathway
Abstract
The invention provides cell-permeable peptides that bind to JNK
proteins and inhibit JNK-mediated effects in JNK-expressing
cells.
Inventors: |
Bonny, Christophe; (Morges,
CH) |
Correspondence
Address: |
MINTZ, LEVIN, COHN, FERRIS, GLOVSKY
AND POPEO, P.C.
ONE FINANCIAL CENTER
BOSTON
MA
02111
US
|
Family ID: |
46299396 |
Appl. No.: |
10/457614 |
Filed: |
June 9, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10457614 |
Jun 9, 2003 |
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10165250 |
Jun 7, 2002 |
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10457614 |
Jun 9, 2003 |
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09503954 |
Feb 14, 2000 |
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6610820 |
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60347062 |
Jan 9, 2002 |
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60158774 |
Oct 12, 1999 |
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Current U.S.
Class: |
514/15.1 ;
514/17.8; 514/17.9; 514/18.2; 514/18.9; 514/3.8 |
Current CPC
Class: |
A61P 25/28 20180101;
A61P 37/02 20180101; A61P 25/00 20180101; A61P 17/14 20180101; C07K
14/4703 20130101; C07K 2319/00 20130101; A61K 38/1709 20130101;
C07K 14/00 20130101 |
Class at
Publication: |
514/012 |
International
Class: |
A61K 038/17 |
Claims
What is claimed is:
1. A method of inhibiting neuronal cell damage in a subject,
comprising administering to the subject prior to identification of
said cell damage a composition comprising a peptide selected from
the group consisting of the amino acid sequence of SEQ ID NO: 1-6,
11-16 and 23-26.
2. The method of claim 1, wherein the peptide comprises the amino
acid sequence of SEQ ID NO: 23.
3. The method of claim 1, wherein the peptide comprises the amino
acid sequence of SEQ ID NO: 24.
4. The method of claim 1, wherein the subject is at risk of
developing a condition characterized by aberrant cell damage.
5. The method of claim 4, wherein said aberrant cell damage is
excitotoxic cell death.
6. The method of claim 4, wherein said aberrant cell damage is
apoptotic cell death.
7. The method of claim 1, wherein said subject is at risk of
developing stroke, amyotrophic lateral sclerosis, epilepsy,
multiple sclerosis, Alzheimer's disease, Parkinson's disease,
Huntington's disease, neurolathyrism, human immunodeficiency virus
dementia, or autoimmune disease.
8. The method of claim 7, wherein said stroke is ischemic
stroke.
9. A method of inhibiting an ischemic or reperfusion related injury
in a subject, comprising administering to the subject prior to
identification of symptom of ischemia or reperfusion injury a
comprising stroke, amyotrophic lateral sclerosis, epilepsy,
multiple sclerosis, Alzheimer's disease, Parkinson's disease,
Huntington's disease, neurolathyrism, human immunodeficiency virus
dementia, or autoimmune disease.
10. A method of inhibiting neuronal cell death in a mammal,
comprising administering to said mammal comprising a peptide
selected from the group consisting of the amino acid sequence of
SEQ ID NO: 1-6, 11-16 and 23-26, wherein said mammal is suffering
from or at risk of developing a neuronal disorder.
11. The method of claim 10, wherein the peptide comprises the amino
acid sequence of SEQ ID NO: 23.
12. The method of claim 10, wherein the peptide comprises the amino
acid sequence of SEQ ID NO: 24.
13. The method of claim 10, wherein said neuronal disorder is
selected from the group consisting of stroke, amyotrophic lateral
sclerosis, epilepsy, multiple sclerosis, Alzheimer's disease,
Parkinson's disease, Huntington's disease, neurolathyrism, and
human immunodeficiency virus dementia.
14. A method of treating a neuronal disorder, comprising
administering to said mammal a composition comprising a peptide
selected from the group consisting of the amino acid sequence of
SEQ ID NO: 1-6, 11-16 and 23-26.
15. The method of claim 14, wherein the peptide comprises the amino
acid sequence of SEQ ID NO: 23.
16. The method of claim 14, wherein the peptide comprises the amino
acid sequence of SEQ ID NO: 24.
17. The method of claim 14, wherein said neuronal disorder is
selected from the group consisting of stroke, amyotrophic lateral
sclerosis, epilepsy, multiple sclerosis, Alzheimer's disease,
Parkinson's disease, Huntington's disease, neurolathyrism, and
human immunodeficiency virus dementia.
18. A method of inhibiting neuronal cell death, comprising
contacting said cell with a composition comprising a peptide
selected from the group consisting of SEQ ID NO: 1-6, 11-16 and
23-26.
19. The method of claim 18, wherein the peptide comprises the amino
acid sequence of SEQ ID NO: 23.
20. The method of claim 18, wherein the peptide comprises the amino
acid sequence of SEQ ID NO: 24.
21. The method of claim 18, wherein said cell death is excitotoxic
cell death.
22. The method of claim 18, wherein said cell death is apoptotic
cell death.
Description
RELATED U.S. APPLICATION
[0001] This application is a continuation-in-part of U.S. Ser. No.
10/165,250, filed Jun. 7, 2002, which claims the benefit of U.S. S
No. 60/347,062, filed Jan. 9, 2002; and to U.S. Ser. No.
09/503,954, filed, Feb. 14, 2000, which claims the benefit of U.S.
S No. 60/158,774, filed on Oct. 12, 1999, each of which is
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] This invention relates generally to protein kinase
inhibitors and more specifically to inhibitors of the protein
kinase c-Jun amino terminal kinase.
BACKGROUND OF THE INVENTION
[0003] The c-Jun amino terminal kinase (JNK) is a member of the
stress-activated group of mitogen-activated protein (MAP) kinases.
These kinases have been implicated in the control of cell growth
and differentiation, and, more generally, in the response of cells
to environmental stimuli. The JNK signal transduction pathway is
activated in response to environmental stress and by the engagement
of several classes of cell surface receptors. These receptors can
include cytokine receptors, serpentine receptors, and receptor
tyrosine kinases. In mammalian cells, JNK has been implicated in
such biological processes as oncogenic transformation and in
mediating adaptive responses to environmental stress. JNK has also
been associated with modulating immune responses, including
maturation and differentiation of immune cells, as well effecting
programmed cell death in cells identified for destruction by the
immune system.
SUMMARY OF THE INVENTION
[0004] The present invention is based in part on the discovery of
peptides that are effective inhibitors of JNK proteins. The
peptides, referred to herein as JNK peptide inhibitors, decrease
the downstream cell-proliferative effects of c-Jun amino terminal
kinase (JNK).
[0005] Accordingly, the invention includes novel JNK inhibitor
peptides ("JNKI peptides"), as well as chimeric peptides which
include a JNK peptide inhibitor linked a trafficking peptide that
can be used to direct a peptide on which it is present do a desired
cellular location. The trafficking sequence can be used to direct
transport of the peptide across the plasma membrane. Alternatively,
or in addition, the trafficking peptide can be used to direct the
peptide to desired intracellular location, such as the nucleus.
[0006] The JNK inhibitor peptides can be present as polymers of
L-amino acids. Alternatively, the peptides can be present as
polymers of D-amino acids.
[0007] Also included in the invention are pharmaceutical
compositions that include the JNK-binding peptides, as well as
antibodies that specifically recognize the JNK-binding
peptides.
[0008] The invention also includes a method of inhibiting
expression of a JNK kinase in a cell.
[0009] In another aspect, the invention includes a method of
treating a pathophysiology associated with activation of JNK in a
cell or cells. For example, the target cells may be, e.g., cultured
animal cells, human cells or micro-organisms. Delivery can be
carried out in vivo by administering the chimeric peptide to an
individual in whom it is to be used for diagnostic, preventative or
therapeutic purposes. The target cells may be in vivo cells, i.e.,
cells composing the organs or tissues of living animals or humans,
or microorganisms found in living animals or humans.
[0010] The invention further provides a method of preventing or
treating hearing loss in a subject. The method includes
administering to the subject a cell-permeable bioactive peptide
which prevents damage to the hair cell stereocilia, hair cell
apoptosis, or neuronal apoptosis. A cell-permeable bioactive
peptide is, for example, a JNK-inhibitor peptide. Preferably, the
cell-permeable bioactive peptide is SEQ ID NOs:1, 2, 4, 5, 6, 11,
12, 13, 14, 15, or 16.
[0011] The hearing loss is caused by a noise trauma. Thus, in one
aspect, the peptide is administered before the subject is exposed
to a noise trauma. In another aspect, the peptide is administered
after the subject is exposed to a noise trauma. The noise trauma
can be, e.g., at least 90 dB SPL. Alternatively, the hearing loss
is caused by antibiotic treatment. Thus, in one aspect, the peptide
is administered before the subject is exposed to an antibiotic. In
another aspect, the peptide is administered after the subject is
exposed to an antibiotic. The antibiotic is, e.g., an
aminoglycoside.
[0012] The hearing loss is caused by a chemotherapeutic agent.
Thus, in one aspect, the peptide is administered before the subject
is exposed to a chemotherapeutic agent. In another aspect, the
peptide is administered after the subject is exposed to a
chemotherapeutic agent.
[0013] The invention further provides a method of preventing or
treating neuronal death or brain lesions in a subject. The method
includes administering to the subject a cell-permeable bioactive
peptide which prevents damage to the neurons or neuronal apoptosis.
A cell-permeable bioactive peptide is, for example, a JNK-inhibitor
("JNKI") peptide. Preferably, the cell-permeable bioactive peptide
is SEQ ID NOs:1, 2, 3, 4, 5, 6, 11, 12, 13, 14, 15, 16, or
21-28.
[0014] The neuronal death or brain lesions are caused by cerebral
ischemia. Thus, in one aspect, the peptide is administered before
the subject experiences an ischemic event. In another aspect, the
peptide is administered after the subject experiences an ischemic
event. The ischemic event is e, e.g., chronic or acute.
[0015] The neuronal death or brain lesions are caused by other
excitotoxic mechanisms. Thus, in one aspect, the peptide is
administered before the subject experiences an excitotoxic
mechanism. In another aspect, the peptide is administered after the
subject experiences an excitotoxic mechanism. The excitotoxic
mechanism can be, e.g., hypoxic/ischemic brain damage, traumatic
brain damages, neuronal death arising from epileptic seizures, and
several neurodegenerative disorders, such as Alzheimer's
disease.
[0016] The invention also contemplates a method of inhibiting
pancreatic islet cell death, where the method includes contacting a
pancreatic islet cell with a cell-permeable bioactive peptide such
that pancreatic cell death is inhibited. A cell-permeable bioactive
peptide is, for example, a JNK-inhibitor peptide. Preferably, the
cell-permeable bioactive peptide is SEQ ID NOs:1, 2, 3, 4, 5, 6,
11, 12, 13, 14, 15, 16, or 21-28. The method can further include
contacting the cell with collagenase.
[0017] Additionally, the invention contemplates a method of
inhibiting pancreatic islet cell death in a subject by
administering to the subject a cell-permeable bioactive peptide
such that pancreatic cell death is inhibited. A cell-permeable
bioactive peptide is, for example, a JNK-inhibitor peptide.
Preferably, the cell-permeable bioactive peptide is SEQ ID NOs:1,
2, 3, 4, 5, 6, 11, 12, 13, 14, 15, 16, or 21-28. The method can
further include contacting the cell with collagenase. In one
embodiment, the cell-permeable bioactive peptide is administered
before the subject is exposed to a pro-inflammatory cytokine. In
another embodiment, the cell-permeable bioactive peptide is
administered after the subject is exposed to a pro-inflammatory
cytokine
[0018] In some aspects, the administration of the peptides of the
invention can be by any one administration route selected from:
intrauricular; intraperitoneal, nasal, intravenous, oral and patch
delivery.
[0019] Among the advantages provided by the invention is that the
JNK inhibitor peptides are small, and can be produced readily in
bulk quantities and in high purity. The inhibitor peptides are also
resistant to intracellular degradation, and are weakly immunogenic.
Accordingly, the peptides are well suited for in vitro and in vivo
applications in which inhibition of JNK-expression is desired.
[0020] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In the case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0021] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIGS. 1A-C are diagrams showing alignments of conserved JBD
domain regions in the indicated transcription factors.
[0023] FIG. 2 is a diagram showing alignments of generic TAT-IB
fusion peptides.
[0024] FIG. 3 is a histogram depicting inhibition of P-cell death
by the minimal 23 amino acid long JBD domain of IB1 compared to the
full 280 amino acid JBD domain.
[0025] FIG. 4 is an illustration demonstrating the effects of TAT,
TAT-IB1 and TAT-IB2 peptides on phosphorylation of recombinant
JNKs. Panel A shows inhibition of c-Jun, ATF2 and Elk1
phosphorylation by recombinant JNKs in vitro. Panel B shows dose
response experiments similar to Panel A.
[0026] FIG. 5 is a histogram depicting L-TAT-IB inhibition of
phosphorylation by recombinant JNKs. Panel A shows L-TAT-IB
inhibition of c-Jun, ATF2 and Elk1 phosphorylation by recombinant
JNKs in vitro in the presence of MKK4. Panel B shows similar dose
response experiments with MKK7.
[0027] FIG. 6 is an illustration demonstrating the inhibition of
c-Jun phosphorylation by activated JNKs.
[0028] FIG. 7 is a histogram depicting short term inhibition of
IL-1.beta. induced pancreatic .beta.-cell death by the L-TAT-IB
peptides.
[0029] FIG. 8 is a histogram depicting short term inhibition of
IL-1.beta. induced pancreatic .beta.-cell death by the D-TAT-IB
peptides.
[0030] FIG. 9 is a histogram depicting long term inhibition of
IL-1.beta. induced pancreatic .beta.-cell death by L-TAT-IB 1 and
D-TAT-IB 1 peptides.
[0031] FIG. 10 is a histogram depicting inhibition of irradiation
induced human colon cancer WiDr cell death by L-TAT-IB1 and
D-TAT-IB1 peptides.
[0032] FIG. 11 is an illustration showing the modulation of JNK
kinase activity by L-TAT, TAT-IB1 and D-TAT-IB1 peptides.
[0033] FIG. 12 are graphs depicting the protective effects of the
TAT-IB1 peptides in mice. Panel A shows the effect of irradiation
on weight. Panel B shows the effect of irradiation on oedemus and
erythemus status.
[0034] FIG. 13 is a figure depicting the protective effect of
D-JNK1 on noise-induced hearing loss. Panel A shows a schematic
depiction of the experiment, panel B shows a graph of hearing loss,
panels C and D depict histological examination of the contralateral
(control) and D-JNK1-injected ear, respectively.
[0035] FIGS. 14A and B are figures depicting the protective effect
of D-JNK1 on antibiotic-induced hearing loss.
[0036] FIG. 15 is a bar graph depicting the increased recovery of
pancreatic islets subjected to D-JNK1 treatment during the
isolation procedure.
[0037] FIG. 16A is an illustration demonstrating the sensitivity
and specificity of the JNK-inhibitory (JNKI) peptides of the
present invention against JNK activation and action. FIG. 16A
demonstrates the inhibitory effect of L-JNKI1 and D-JNKI1 on JNK
activation and action in kinase assays with recombinant
JNK1.alpha.1 and GST-Jun and GST-Elk1 substrates, respectively.
[0038] FIG. 16B is an illustration demonstrating the sensitivity
and specificity of the JNK-inhibitory (JNKI) peptides of the
present invention against JNK activation and action. FIG. 16B
demonstrates the inhibitory effect of the 20 amino acid minimal
JNK-inhibitory sequence of JIP-IB1 (L-form of JBD.sub.20) in dose
response experiments, using conditions similar to those in FIG. 16A
and with decreasing amounts of L-JBD.sub.20.
[0039] FIG. 16C is an illustration demonstrating the sensitivity
and specificity of the JNK-inhibitory (JNKI) peptides of the
present invention against JNK activation and action. FIG. 16C
demonstrates the specificity of the JNKI peptides of the present
invention in blocking JNK activation using kinase assays with
different recombinant kinases.
[0040] FIG. 17A is an illustration demonstrating
N-methyl-D-aspartate ("NMDA")-induced activation of JNK in
untreated neurons (0) and in neurons exposed to 100 .mu.M NMDA for
10 minutes (10') or for 30 minutes (30').
[0041] FIG. 17B is an illustration that demonstrates the effects of
the JNKI peptides of the present invention on the level of c-Jun
phosphorylation and the amount of JNK after exposure to NMDA. In
FIG. 17B, 4-fold more protein was loaded in the nuclear extracts
(Nucl) than in the cytoplasmic ones (Cyt), and the abbreviations
used are: C: Control; N: NMDA; L: L-JNI1+NMDA; D: D-JNKI1+NMDA.
[0042] FIG. 17C is a histogram that depicts the quantification of
c-fos expression by real-time PCR using extracted RNA. FIG. 17C
illustrates the expression of c-fos relative to actin.
[0043] FIG. 18 are illustrations and a histogram depicting the time
course of NMDA neurotoxicity and neuroprotection by L-JNKI1,
D-JNKI1 and two control peptides, TAT-empty (the TAT sequence
alone, without JBD.sub.20) and L-JNKI1-mut (wherein 6 amino acids
have been mutated to alanine).
[0044] FIGS. 18A-18E are a series of micrographs that depict
Hoechst-stained neurons at 24 hours after NMDA treatment.
[0045] FIG. 18F is a histogram depicting neuronal death at 12 h, 24
h, and 48 h after NMDA exposure (100 .mu.M NMDA), as indicated by
LDH activity.
[0046] FIG. 19 are illustrations and a histogram depicting
transient ischemia in mice.
[0047] FIG. 19A demonstrates the effect on infarct volume of a
pretreatment in which an intracerebro-ventricular (icv) injection
of D-JNKI1 (15.7 ng in 2 .mu.L phosphate buffer solution (PBS)) was
administered to a subject 1 hour prior to occlusion.
[0048] FIG. 19B demonstrates the effect on infarct volume where the
icv injection of D-JKNI1 was administered 1 hour prior to occlusion
or at 3 hours, 6 hours and 12 hours post-occlusion.
[0049] FIG. 20 are illustrations and a histogram demonstrating
protection by D-JNKI1 against permanent focal ischemia in young
rats (P14) that had been perfused 24 hours post-occlusion.
[0050] FIG. 20A is a series of illustrations that depicts examples
of lesions from a control rat (left panel) and a rat treated with
D-JNKI6 hours after occlusion (right panel).
[0051] FIG. 20B is a histogram depicting the infarct volumes,
expressed as % of hemispheric volume, following the
intra-peritoneal (i.p.) injection of D-JNKI1 at-0.5 h before or at
+6 h or +12 h after occlusion.
[0052] FIG. 20C is a series of illustrations depicting the results
of the immunohistochemistry for P-c-Jun in which c-Jun was
phosphorylated in many neurons in the peri-infarcted cortex.
DETAILED DESCRIPTION OF THE INVENTION
[0053] The present invention is based in part on the discovery of
cell permeable peptides that inhibit the activated c-Jun amino
terminal kinase (JNK) signaling pathway. These peptides are
referred to herein as JNK inhibitor peptides. Additionally, the
discovery provides methods and pharmaceutical compositions for
treating pathophysiologies associated with JNK signaling.
[0054] JNK inhibitor peptides were identified by inspecting
sequence alignments between kJNK Binding Domains in various insulin
binding (IB) proteins. The results of this alignment are shown in
FIGS. 1A-1C. FIG. 1A depicts the region of highest homology between
the JBDs of IB1, IB2, c-Jun and ATF2. Panel B depicts the amino
acid sequence alignment of the JBDs of IB1 and IB2. Fully conserved
residues are indicated by asterisks, while residues changed to Ala
in the GFP-JBD.sub.23Mut vector are indicated by open circles. FIG.
1C shows the amino acid sequences of chimeric proteins that include
a JNK inhibitor peptide domain and a trafficking domain. In the
example shown, the trafficking domain is derived from the human
immunodeficiency virus (HIV) TAT polypeptide, and the JNK inhibitor
peptide is derived from an IB1 polypeptide. Human, mouse, and rat
sequences are identical in Panels B and C.
[0055] Sequence comparison between the JNK binding domains of IB1
[SEQ ID NO: 17], IB2 [SEQ ID NO: 18], c-Jun [SEQ ID NO: 19] and
ATF2 [SEQ ID NO: 20] revealed a partially conserved 8 amino acid
sequence (FIG. 1A). A comparison of the JBDs of IB1 and IB2 further
revealed two blocks of seven and three amino acids that are highly
conserved between the two sequences. These two blocks are contained
within a peptide sequence of 23 amino acids in IB1 [SEQ ID NO: 1]
and 21 amino acids in IB2 [SEQ ID NO: 2]. The 20 amino acid minimal
JNK-inhibitory sequence of JIP-IB1 (L-form of JBD.sub.20 (SEQ ID
NO:21)) is shown in FIG. 1C.
[0056] The JNK inhibitor peptides of the invention can be used in
any situation in which inhibition of JNK activity is desired. This
can include in vitro applications, ex vivo, and in vivo
applications. As JNKs and all its isoforms participate in the
development and establishment of pathological states or in
pathways, the JNK peptides can be used to prevent or inhibit the
occurrence of such pathological states. This includes prevention
and treatment of diseases and prevention and treatment of
conditions secondary to therapeutic actions. For example, the
peptides of the present invention can be used to treat or prevent,
e.g., diabetes, ionizing radiation, immune responses (including
autoimmune diseases), ischemia/reperfusion injuries, heart and
cardiovascular hypertrophies, and some cancers (e.g., Bcr-Abl
transformation).
[0057] The peptides can also be used to inhibit expression of genes
whose expression increases in the presence of an active JNK
polypeptide. These genes and gene products includes, e.g.,
proinflammatory cytokines. Such cytokines are found in all forms of
inflammatory, auto-inflammatory, immune and autoimmune diseases,
degenerative diseases, myopathies, cardiomyopathies, and graft
rejection.
[0058] The JNK inhibitor peptides described herein can also be used
to treat or prevent effects associated with cellular shear stress,
such as in pathological states induced by arterial hypertension,
including cardiac hypertrophy and arteriosclerotic lesions, and at
bifurcations of blood vessels, and the like; ionizing radiation, as
used in radiotherapy and UV lights; free radicals; DNA damaging
agents, including chemotherapeutic drugs; oncogenic transformation;
neuronal and pancreatic cell damage, hearing loss, ischemia and
reperfusion; hypoxia; and hypo- and hyperthermia.
[0059] The polynucleotides provided by the present invention can be
used to express recombinant peptides for analysis, characterization
or therapeutic use; as markers for tissues in which the
corresponding peptides is preferentially expressed (either
constitutively or at a particular stage of tissue differentiation
or development or in disease states). Other uses for the nucleic
acids include, e.g., molecular weight markers in gel
electrophoresis-based analysis of nucleic acids.
[0060] The JNK inhibitor peptides disclosed herein are presented in
Table 1. The table presents the name of the JNK inhibitor peptide,
as well as its sequence identifier number, length, and amino acid
sequence.
1TABLE 1 PEPTIDE NAME SEQ ID AA Sequence L-IB1 1 23 DTYRPKRPTT
LNLFPQVPRS QDT L-IB2 2 21 EEPHKHRPTT LRLTTLGAQD S D-IB1 3 23
TDQSRPVQPF LNLTTPRKPR YTD D-IB2 4 21 SDQAGLTTLR LTTPRHKHPE E L-IB
(generic) 5 19 XRPTTLXLXX XXXXXQDS/TX D-IB (generic) 6 19
XS/TDQXXXXXX XLXLTTPRX L-TAT 7 10 GRKKRRQRRR D-TAT 8 10 RRRQRRKKRG
L-generic-TAT 9 17 XXXXRKKRRQ RRRXXXX D-generic-TAT 10 17
XXXXRRRQRR KKRXXXX L-TAT-IB1 11 35 GRKKRRQRRR PPDTYRPKRP TTLNLFPQVP
RSQDT L-TAT-IB2 12 33 GRKKRRQRRR PPEEPHKHRP TTLRLTTLGA QDS L-TAT-IB
(generic) 13 42 XXXXXXXRKK RRQRRRXXXX XXXXRPTTLX LXXXXXXXQD S/TX
D-TAT-IB1 14 35 TDQSRPVQPF LNLTTPRKPR YTDPPRRRQR RKKRG D-TAT-IB2 15
33 SDQAGLTTLR LTTPRHKHPE EPPRRRQRRK KRG D-TAT-IB (generic) 16 42
XT/SDQXXXXXX XLXLTTPRXX XXXXXXRRRQ RRKKRXXXXX XX IB1-long 17 29
PGTGCGDTYR PKRPTTLNLF PQVPRSQDT IB2-long 18 27 IPSPSVEEPH
KHRPTTLRLT TLGAQDS c-Jun 19 29 GAYGYSNPKI LKQSMTLNLA DPVGNLKPH ATF2
20 29 TNEDHLAVHK HKHEMTLKFG PARNDSVIV L-JBD.sub.20 21 20 RPKRPTTLNL
FPQVPRSQDT D-JBD.sub.20 22 20 TDQSRPVQPF LNLTTPRKPR L-TAT-JNKI1
(i.e., 23 32 GRKKRRQRRR PPRPKRPTTL NLFPQVPRSQ DT L-TAT-JBD.sub.20)
D-TAT-JNKI1 (i.e., 24 32 TDQSRPVQPF LNLTTPRKPR YTDPPRRRQR RKKRG
D-TAT-JBD.sub.20) L-TAT-JNKI1 25 34 XXXXRKKRRQ RRRXXXXRPT
TLXLXXXXXX XQDS/T (generic) D-TAT-JNKI1 26 34 S/TDQXXXXXXX
LXLTTPRXXX XRRRQRRKKR XXXX (generic) L-JBD.sub.20-mut 27 20
RPKRPTAANA FPQVPRSQDT D-JBD.sub.20-mut 28 20 TDQSRPVAPF
ANAATPRKPR
[0061] JNK Inhibitor Peptides
[0062] In one aspect, the invention provides a JNK inhibitor
peptide. No particular length is implied by the term "peptide." In
some embodiments, the JNK-inhibitor peptide is less than 280 amino
acids in length, e.g., less than or equal to 150, 100, 75, 50, 35,
or 25 amino acids in length. In various embodiment, the JNK-binding
inhibitor peptide includes the amino acid sequence of one or more
of SEQ ID NOs: 1-6 and 21-22. In one embodiment, the JNK inhibitor
peptide peptides bind JNK. In another embodiment the peptide
inhibits the activation of at least one JNK activated transcription
factor, e.g. c-Jun, ATF2 or Elk1.
[0063] Examples of JNK inhibitor peptides include a peptide which
includes (in whole or in part) the sequence
NH.sub.2-DTYRPKRPTTLNLFPQVPRSQDT-COOH [SEQ ID NO:1]. In another
embodiment, the peptide includes the sequence
NH.sub.2-EEPHKHRPTTLRLTTLGAQDS-COOH [SEQ ID NO:2] Alternatively,
examples of JNK inhibitor peptides include a peptide which includes
(in whole or in part) the sequence NH.sub.2-RPKRPTTLNL
FPQVPRSQDT-COOH [SEQ ID NO:21].
[0064] The JNK inhibitor peptides can be polymers of L-amino acids,
D-amino acids, or a combination of both. For example, in various
embodiments, the peptides are D retro-inverso peptides. The term
"retro-inverso isomer" refers to an isomer of a linear peptide in
which the direction of the sequence is reversed, the term
"D-retro-inverso isomer" refers to an isomer of a linear peptide in
which the direction of the sequence is reversed and the chirality
of each amino acid residue is inverted. See, e.g., Jameson et al.,
Nature, 368, 744-746 (1994); Brady et al., Nature, 368, 692-693
(1994). The net result of combining D-enantiomers and reverse
synthesis is that the positions of carbonyl and amino groups in
each amide bond are exchanged, while the position of the side-chain
groups at each alpha carbon is preserved. Unless specifically
stated otherwise, it is presumed that any given L-amino acid
sequence of the invention may be made into an D retro-inverso
peptide by synthesizing a reverse of the sequence for the
corresponding native L-amino acid sequence.
[0065] For example, a D retro-inverso peptide has the sequence
NH.sub.2-TDQSRPVQPFLNLTTPRKPRYTD-COOH [SEQ ID NO:3] or
NH.sub.2-SDQAGLTTLRLTTPRHKHPEE-COOH [SEQ ID NO: 4]. Alternatively,
a D retro-inverso peptide includes the sequence NH.sub.2-TDQSRPVQPF
LNLTTPRKPR-COOH [SEQ ID NO:22]. It has been unexpectedly found that
D-retro-inverso peptides have a variety of useful properties. For
example, D-TAT and D-TAT-IB and D-TAT-JNKI peptides enter cells as
efficiently as L-TAT and L-TAT-IB and D-TAT-JNKI peptides, and
D-TAT and D-TAT-IB and D-TAT-JNKI peptides are more stable than the
corresponding L-peptides. Further, while D-TAT-IB1 are .about.10-20
fold less efficient in inhibiting JNK than L-TAT-IB and L-TAT-JNKI,
they are 50 fold more stable in vivo. Moreover, the D-retro-inverso
JNKI peptides are protease-resistant. Finally, as is discussed
further below, D-TAT-IB and D-TAT-JNKI peptides protect
interleukin-1 treated and ionizing irradiated cells from apoptosis,
and these peptides are useful in treating neurons, as the TAT
sequence contains six pairs of amino acid that render the TAT
sequence extremely sensitive to the neuronal proteases that are
involved in peptide processing in the nervous system. See e.g.,
Steiner et al., J. Biol. Chem. 267:23435-23438 (1992); Brugidou et
al., Biochem. & Biophys. Res. Comm. 214:685-693 (1995); each of
which is incorporated herein by reference in its entirety.
[0066] A JNK inhibitor peptide according to the invention includes
the amino acid sequence
NH.sub.2-X.sub.n-RPTTLXLXXXXXQDS/T-X.sub.n-COOH [SEQ ID NO: 5, and
residues 17-42 of L-TAT-IB, SEQ ID NO: 13, as shown in FIG. 2]. As
used herein, X.sub.n may be zero residues in length, or may be a
contiguous stretch of peptide residues derived from SEQ ID NOS:1
and 21, preferably a stretch of between 1 and 7 amino acids in
length, or may be 10, 20, 30 or more amino acids in length. The
single residue represented by S/T may be either Ser or Thr in the
generic sequence. In a further embodiment, a JNK inhibitor peptide
of the invention may be a D retro-inverso peptide having the
sequence NH.sub.2-X.sub.n-S/TDQXXXXLXLTT- PR-X.sub.n-COOH [SEQ ID
NO: 6], and residues 17-42 of L-TAT-IB, SEQ ID NO: 16, as shown in
FIG. 2].
[0067] JNK-inhibitor peptides are obtained or produced by methods
well-known in the art, e.g. chemical synthesis, genetic engineering
methods as discussed below. For example, a peptide corresponding to
a portion of a JNK inhibitor peptide including a desired region or
domain, or that mediates the desired activity in vitro, may be
synthesized by use of a peptide synthesizer.
[0068] A candidate JNK inhibitor peptide is analyzed by
hydrophilicity analysis (see, e.g., Hopp and Woods, 1981. Proc Natl
Acad Sci USA 78: 3824-3828) that can be utilized to identify the
hydrophobic and hydrophilic regions of the peptides, thus aiding in
the design of substrates for experimental manipulation, such as in
binding experiments, antibody synthesis. Secondary structural
analysis may also be performed to identify regions of a JNK
inhibitor peptide that assume specific structural motifs. See e.g.,
Chou and Fasman, 1974. Biochem 13: 222-223. Manipulation,
translation, secondary structure prediction, hydrophilicity and
hydrophobicity profiles, open reading frame prediction and
plotting, and determination of sequence homologies can be
accomplished using computer software programs available in the art.
Other methods of structural analysis including, e.g., X-ray
crystallography (see, e.g., Engstrom, 1974. Biochem Exp Biol 11:
7-13); mass spectroscopy and gas chromatography (see, e.g., METHODS
IN PROTEIN SCIENCE, 1997. J. Wiley and Sons, New York, N.Y.) and
computer modeling (see, e.g., Fletterick and Zoller, eds., 1986.
Computer Graphics and Molecular Modeling, In: CURRENT
COMMUNICATIONS IN MOLECULAR BIOLOGY, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y.) may also be employed.
[0069] The present invention additionally relates to nucleic acids
that encode JNK-binding peptides having L-form amino acids, e.g.,
those L-peptides indicated in Table 1, as well as the complements
of these sequences. Suitable sources of nucleic acids encoding JNK
inhibitor peptides include the human IB1 nucleic acid (and the
encoded protein sequences) available as GenBank Accession Nos.
AF074091 and AAD20443, respectively. Other sources include rat IB1
nucleic acid and protein sequences are shown in GenBank Accession
No. AF108959 and AAD22543, respectively, and are incorporated
herein by reference in their entirety. Human IB2 nucleic acid and
protein sequences are shown in GenBank Accession No AF218778 and is
also incorporated herein by reference in their entirety.
[0070] Nucleic acids encoding the JNK inhibitor peptides may be
obtained by any method known in the art (e.g., by PCR amplification
using synthetic primers hybridizable to the 3'- and 5'-termini of
the sequence and/or by cloning from a cDNA or genomic library using
an oligonucleotide sequence specific for the given gene
sequence).
[0071] For recombinant expression of one or more JNK inhibitor
peptides, the nucleic acid containing all or a portion of the
nucleotide sequence encoding the peptide may be inserted into an
appropriate expression vector (i.e., a vector that contains the
necessary elements for the transcription and translation of the
inserted peptide coding sequence). In some embodiments, the
regulatory elements are heterologous (i.e., not the native gene
promoter). Alternately, the necessary transcriptional and
translational signals may also be supplied by the native promoter
for the genes and/or their flanking regions.
[0072] A variety of host-vector systems may be utilized to express
the peptide coding sequence(s). These include, but are not limited
to: (i) mammalian cell systems that are infected with vaccinia
virus, adenovirus, and the like; (ii) insect cell systems infected
with baculovirus and the like; (iii) yeast containing yeast vectors
or (iv) bacteria transformed with bacteriophage, DNA, plasmid DNA,
or cosmid DNA. Depending upon the host-vector system utilized, any
one of a number of suitable transcription and translation elements
may be used.
[0073] Promoter/enhancer sequences within expression vectors may
utilize plant, animal, insect, or fungus regulatory sequences, as
provided in the invention. For example, promoter/enhancer elements
can b used from yeast and other fungi (e.g., the GAL4 promoter, the
alcohol dehydrogenase promoter, the phosphoglycerol kinase
promoter, the alkaline phosphatase promoter). Alternatively, or in
addition, they may include animal transcriptional control regions,
e.g., (i) the insulin gene control region active within pancreatic
.beta.-cells (see, e.g., Hanahan, et al., 1985. Nature 315:
115-122); (ii) the immunoglobulin gene control region active within
lymphoid cells (see, e.g., Grosschedl, et al., 1984. Cell 38:
647-658); (iii) the albumin gene control region active within liver
(see, e.g., Pinckert, et al., 1987. Genes and Dev 1: 268-276; (iv)
the myelin basic protein gene control region active within brain
oligodendrocyte cells (see, e.g., Readhead, et al., 1987. Cell 48:
703-712); and (v) the gonadotropin-releasing hormone gene control
region active within the hypothalamus (see, e.g., Mason, et al.,
1986. Science 234: 1372-1378), and the like.
[0074] Expression vectors or their derivatives include, e.g. human
or animal viruses (e.g., vaccinia virus or adenovirus); insect
viruses (e.g., baculovirus); yeast vectors; bacteriophage vectors
(e.g., lambda phage); plasmid vectors and cosmid vectors.
[0075] A host cell strain may be selected that modulates the
expression of inserted sequences of interest, or modifies or
processes expressed peptides encoded by the sequences in the
specific manner desired. In addition, expression from certain
promoters may be enhanced in the presence of certain inducers in a
selected host strain; thus facilitating control of the expression
of a genetically-engineered peptides. Moreover, different host
cells possess characteristic and specific mechanisms for the
translational and post-translational processing and modification
(e.g., glycosylation, phosphorylation, and the like) of expressed
peptides. Appropriate cell lines or host systems may thus be chosen
to ensure the desired modification and processing of the foreign
peptide is achieved. For example, peptide expression within a
bacterial system can be used to produce an unglycosylated core
peptide; whereas expression within mammalian cells ensures "native"
glycosylation of a heterologous peptide.
[0076] Also included in the invention are derivatives, fragments,
homologs, analogs and variants of JNK inhibitor peptides and
nucleic acids encoding these peptides. For nucleic acids,
derivatives, fragments, and analogs provided herein are defined as
sequences of at least 6 (contiguous) nucleic acids, and which have
a length sufficient to allow for specific hybridization. For amino
acids, derivatives, fragments, and analogs provided herein are
defined as sequences of at least 4 (contiguous) amino acids, a
length sufficient to allow for specific recognition of an
epitope.
[0077] The length of the fragments are less than the length of the
corresponding full-length nucleic acid or polypeptide from which
the JNK inhibitor peptide, or nucleic acid encoding same, is
derived. Derivatives and analogs may be full length or other than
full length, if the derivative or analog contains a modified
nucleic acid or amino acid. Derivatives or analogs of the JNK
inhibitor peptides include, e.g., molecules including regions that
are substantially homologous to the peptides, in various
embodiments, by at least about 30%, 50%, 70%, 80%, or 95%, 98%, or
even 99%, identity over an amino acid sequence of identical size or
when compared to an aligned sequence in which the alignment is done
by a computer homology program known in the art. For example
sequence identity can be measured using sequence analysis software
(Sequence Analysis Software Package of the Genetics Computer Group,
University of Wisconsin Biotechnology Center, 1710 University
Avenue, Madison, Wis. 53705), with the default parameters
therein.
[0078] In the case of polypeptide sequences, which are less than
100% identical to a reference sequence, the non-identical positions
are preferably, but not necessarily, conservative substitutions for
the reference sequence. Conservative substitutions typically
include substitutions within the following groups: glycine and
alanine; valine, isoleucine, and leucine; aspartic acid and
glutamic acid; asparagine and glutamine; serine and threonine;
lysine and arginine; and phenylalanine and tyrosine. Thus, included
in the invention are peptides having mutated sequences such that
they remain homologous, e.g. in sequence, in function, and in
antigenic character or other function, with a protein having the
corresponding parent sequence. Such mutations can, for example, be
mutations involving conservative amino acid changes, e.g., changes
between amino acids of broadly similar molecular properties. For
example, interchanges within the aliphatic group alanine, valine,
leucine and isoleucine can be considered as conservative. Sometimes
substitution of glycine for one of these can also be considered
conservative. Other conservative interchanges include those within
the aliphatic group aspartate and glutamate; within the amide group
asparagine and glutamine; within the hydroxyl group serine and
threonine; within the aromatic group phenylalanine, tyrosine and
tryptophan; within the basic group lysine, arginine and histidine;
and within the sulfur-containing group methionine and cysteine.
Sometimes substitution within the group methionine and leucine can
also be considered conservative. Preferred conservative
substitution groups are aspartate-glutamate; asparagine-glutamine;
valine-leucine-isoleucine; alanine-valine; phenylalanine-tyrosine;
and lysine-arginine.
[0079] Where a particular polypeptide is said to have a specific
percent identity to a reference polypeptide of a defined length,
the percent identity is relative to the reference peptide. Thus, a
peptide that is 50% identical to a reference polypeptide that is
100 amino acids long can be a 50 amino acid polypeptide that is
completely identical to a 50 amino acid long portion of the
reference polypeptide. It might also be a 100 amino acid long
polypeptide, which is 50% identical to the reference polypeptide
over its entire length. Of course, other polypeptides will meet the
same criteria.
[0080] The invention also encompasses allelic variants of the
disclosed polynucleotides or peptides; that is, naturally-occurring
alternative forms of the isolated polynucleotide that also encode
peptides that are identical, homologous or related to that encoded
by the polynucleotides. Alternatively, non-naturally occurring
variants may be produced by mutagenesis techniques or by direct
synthesis.
[0081] Species homologs of the disclosed polynucleotides and
peptides are also provided by the present invention. "Variant"
refers to a polynucleotide or polypeptide differing from the
polynucleotide or polypeptide of the present invention, but
retaining essential properties thereof. Generally, variants are
overall closely similar, and in many regions, identical to the
polynucleotide or polypeptide of the present invention. The
variants may contain alterations in the coding regions, non-coding
regions, or both.
[0082] In some embodiments, altered sequences include insertions
such that the overall amino acid sequence is lengthened while the
protein retains trafficking properties. Additionally, altered
sequences may include random or designed internal deletions that
shorten the overall amino acid sequence while the protein retains
transport properties.
[0083] The altered sequences can additionally or alternatively be
encoded by polynucleotides that hybridize under stringent
conditions with the appropriate strand of the naturally-occurring
polynucleotide encoding a polypeptide or peptide from which the JNK
inhibitor peptide is derived. The variant peptide can be tested for
JNK-binding and modulation of JNK-mediated activity using the
herein described assays. `Stringent conditions` are sequence
dependent and will be different in different circumstances.
Generally, stringent conditions can be selected to be about
5.degree. C. lower than the thermal melting point (T.sub.M) for the
specific sequence at a defined ionic strength and pH. The T.sub.M
is the temperature (under defined ionic strength and pH) at which
50% of the target sequence hybridizes to a perfectly matched probe.
Typically, stringent conditions will be those in which the salt
concentration is at least about 0.02 molar at pH 7 and the
temperature is at least about 60.degree. C. As other factors may
affect the stringency of hybridization (including, among others,
base composition and size of the complementary strands), the
presence of organic solvents and the extent of base mismatching,
the combination of parameters is more important than the absolute
measure of any one.
[0084] High stringency can include, e.g., Step 1: Filters
containing DNA are pretreated for 8 hours to overnight at
65.degree. C. in buffer composed of 6.times.SSC, 50 mM Tris-HCl (pH
7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500
.mu.g/ml denatured salmon sperm DNA. Step 2: Filters are hybridized
for 48 hours at 65.degree. C. in the above prehybridization mixture
to which is added 100 mg/ml denatured salmon sperm DNA and
5-20.times.10.sup.6 cpm of .sup.32P-labeled probe. Step 3: Filters
are washed for 1 hour at 37.degree. C. in a solution containing
2.times.SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is
followed by a wash in 0.1.times.SSC at 50.degree. C. for 45
minutes. Step 4: Filters are autoradiographed. Other conditions of
high stringency that may be used are well known in the art. See,
e.g., Ausubel et al., (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR
BIOLOGY, John Wiley and Sons, NY; and Kriegler, 1990, GENE TRANSFER
AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY.
[0085] Moderate stringency conditions can include the following:
Step 1: Filters containing DNA are pretreated for 6 hours at
55.degree. C. in a solution containing 6.times.SSC, 5.times.
Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm
DNA. Step 2: Filters are hybridized for 18-20 hours at 55.degree.
C. in the same solution with 5-20.times.106 cpm 32P-labeled probe
added. Step 3: Filters are washed at 37.degree. C. for 1 hour in a
solution containing 2.times.SSC, 0.1% SDS, then washed twice for 30
minutes at 60.degree. C. in a solution containing 1.times.SSC and
0.1% SDS. Step 4: Filters are blotted dry and exposed for
autoradiography. Other conditions of moderate stringency that may
be used are well-known in the art. See, e.g., Ausubel et al.,
(eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley
and Sons, NY; and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A
LABORATORY MANUAL, Stockton Press, NY.
[0086] Low stringency can include: Step 1: Filters containing DNA
are pretreated for 6 hours at 40.degree. C. in a solution
containing 35% formamide, 5.times.SSC, 50 mM Tris-HCl (pH 7.5), 5
mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 .mu.g/ml denatured
salmon sperm DNA. Step 2: Filters are hybridized for 18-20 hours at
40.degree. C. in the same solution with the addition of 0.02% PVP,
0.02% Ficoll, 0.2% BSA, 100 .mu.g/ml salmon sperm DNA, 10% (wt/vol)
dextran sulfate, and 5-20.times.106 cpm .sup.32P-labeled probe.
Step 3: Filters are washed for 1.5 hours at 55.degree. C. in a
solution containing 2.times.SSC, 25 mM Tris-HCl (pH 7.4), 5 mM
EDTA, and 0.1% SDS. The wash solution is replaced with fresh
solution and incubated an additional 1.5 hours at 60.degree. C.
Step 4: Filters are blotted dry and exposed for autoradiography. If
necessary, filters are washed for a third time at 65-68.degree. C.
and reexposed to film. Other conditions of low stringency that may
be used are well known in the art (e.g., as employed for
cross-species hybridizations). See, e.g., Ausubel et al., (eds.),
1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley and Sons,
NY; and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY
MANUAL, Stockton Press, NY.
[0087] Chimeric Peptides Including a JNK Inhibitor Domain and a
Trafficking Domain
[0088] In another aspect the invention provides a chimeric peptide
that includes a first and second domain. The first domain includes
a trafficking sequence, while the second domain includes a JNK
inhibitor sequence linked by a covalent bond, e.g. peptide bond, to
the first domain. The first and second domains can occur in any
order in the peptide, and the peptide can include one or more of
each domain.
[0089] A trafficking sequence is any sequence of amino acids that
directs a peptide in which it is present to a desired cellular
destination. Thus, the trafficking sequence can direct the peptide
across the plasma membrane, e.g., from outside the cell, through
the plasma membrane, and into the cytoplasm. Alternatively, or in
addition, the trafficking sequence can direct the peptide to a
desired location within the cell, e.g., the nucleus, the ribosome,
the ER, a lysosome, or peroxisome.
[0090] In some embodiments, the trafficking peptide is derived from
a known membrane-translocating sequence. For example, the
trafficking peptide may include sequences from the human
immunodeficiency virus (HIV) 1 TAT protein. This protein is
described in, e.g., U.S. Pat. Nos. 5,804,604 and 5,674,980, each
incorporated herein by reference. The JNK inhibitor peptide is
linked to some or all of the entire 86 amino acids that make up the
TAT protein. For example, a functionally effective fragment or
portion of a TAT protein that has fewer than 86 amino acids, which
exhibits uptake into cells, and optionally uptake into the cell
nucleus, can be used. See e.g., Vives et al., J. Biol. Chem.,
272(25):16010-17 (1997), incorporated herein by reference in its
entirety. In one embodiment, the fragment includes a peptide
containing TAT residues 48-57, e.g. NH.sub.2-GRKKRRQRRR-COOH [SEQ
ID NO: 7] or a generic TAT sequence
NH.sub.2-X.sub.n-RKKRRQRRR-X.sub.n-COOH [SEQ ID NO: 9]. A TAT
peptide that includes the region that mediates entry and uptake
into cells can be further defined using known techniques. See,
e.g., Franked et al., Proc. Natl. Acad. Sci, USA 86: 7397-7401
(1989).
[0091] The TAT sequence may be linked either to the N-terminal or
the C-terminal end of JNK inhibitor sequence. A hinge of two
proline residues may be added between the TAT and JNK inhibitor
peptide to create the full fusion peptide. For example, L-amino
acid fusion peptides may be the L-TAT-IB1 peptide [SEQ ID NO:11],
the L-TAT-IB2 peptide [SEQ ID NO:12], or the generic L-TAT-IB
peptide [SEQ ID NO:13]. Alternatively, L-amino acid fusion peptides
may be the L-TAT-JNKI1 peptide [SEQ ID NO:21] or the generic
L-TAT-JNKI peptide [SEQ ID NO:25]. D retro-inverso fusion peptides
may be the D-TAT-IB1 peptide [SEQ ID NO:14], the DTAT-IB2 peptide
[SEQ ID NO:15], or the generic D-TAT-IB peptide [SEQ ID NO:16].
Alternatively, D retro-inverso fusion peptides may be the
D-TAT-JNKI peptide [SEQ ID NO:22] or the generic D-TAT-JNKI peptide
[SEQ ID NO:26]. The TAT peptide may be a D retro-inverso peptide
having the sequence NH.sub.2-X.sub.n-RRRQRRKKR-X.sub.n-COOH [SEQ ID
NO:10]. In SEQ ID NOs:5-6, 9-10, 13, 16, and 25-26, the number of
"X" residues is not limited to the one depicted and can equal any
number of amino acid residues, including zero, and may vary as
described above.
[0092] The trafficking sequence can be a single (i.e., continuous)
amino acid sequence present in the TAT sequence. Alternatively it
can be two or more amino acid sequences, which are present in TAT
protein, but in the naturally-occurring protein are separated by
other amino acid sequences. As used herein, TAT protein includes a
naturally-occurring amino acid sequence that is the same as that of
naturally-occurring TAT protein, or its functional equivalent
protein or functionally equivalent fragments thereof (peptides).
Such functional equivalent proteins or functionally equivalent
fragments possess uptake activity into the cell and into the cell
nucleus that is substantially similar to that of
naturally-occurring TAT protein. TAT protein can be obtained from
naturally-occurring sources or can be produced using genetic
engineering techniques or chemical synthesis.
[0093] The amino acid sequence of naturally-occurring HIV TAT
protein can be modified, for example, by addition, deletion and/or
substitution of at least one amino acid present in the
naturally-occurring TAT protein, to produce modified TAT protein
(also referred to herein as TAT protein). Modified TAT protein or
TAT peptide analogs with increased or decreased stability can be
produced using known techniques. In some embodiments TAT proteins
or peptides include amino acid sequences that are substantially
similar, although not identical, to that of naturally-occurring TAT
protein or portions thereof. In addition, cholesterol or other
lipid derivatives can be added to TAT protein to produce a modified
TAT having increased membrane solubility.
[0094] Variants of the TAT protein can be designed to modulate
intracellular localization of TAT-JNK inhibitor peptide. When added
exogenously, such variants are designed such that the ability of
TAT to enter cells is retained (i e., the uptake of the variant TAT
protein or peptide into the cell is substantially similar to that
of naturally-occurring HIV TAT). For example, alteration of the
basic region thought to be important for nuclear localization (see,
e.g., Dang and Lee, J. Biol. Chem. 264: 18019-18023 (1989); Hauber
et al., J. Virol. 63: 1181-1187 (1989); Ruben et al., J. Virol. 63:
1-8 (1989)) can result in a cytoplasmic location or partially
cytoplasmic location of TAT, and therefore, of the JNK inhibitor
peptide. Alternatively, a sequence for binding a cytoplasmic or any
other component or compartment (e.g., endoplasmic reticule,
mitochondria, gloom apparatus, lysosomal vesicles,) can be
introduced into TAT in order to retain TAT and the JNK inhibitor
peptide in the cytoplasm or any other compartment to confer
regulation upon uptake of TAT and the JNK inhibitor peptide.
[0095] Other sources for the trafficking peptide include, e.g.,
VP22 (described in, e.g., WO 97/05265; Elliott and O'Hare, Cell 88:
223-233 (1997)), or non-viral proteins (Jackson et al, Proc. Natl.
Acad. Sci. USA 89: 10691-10695 (1992)).
[0096] The JNK inhibitor sequence and the trafficking sequence can
be linked by chemical coupling in any suitable manner known in the
art. Many known chemical cross-linking methods are non-specific,
i.e.; they do not direct the point of coupling to any particular
site on the transport polypeptide or cargo macromolecule. As a
result, use of non-specific cross-linking agents may attack
functional sites or sterically block active sites, rendering the
conjugated proteins biologically inactive.
[0097] One way to increasing coupling specificity is to directly
chemical coupling to a functional group found only once or a few
times in one or both of the polypeptides to be cross-linked. For
example, in many proteins, cysteine, which is the only protein
amino acid containing a thiol group, occurs only a few times. Also,
for example, if a polypeptide contains no lysine residues, a
crosslinking reagent specific for primary amines will be selective
for the amino terminus of that polypeptide. Successful utilization
of this approach to increase coupling specificity requires that the
polypeptide have the suitably rare and reactive residues in areas
of the molecule that may be altered without loss of the molecule's
biological activity.
[0098] Cysteine residues may be replaced when they occur in parts
of a polypeptide sequence where their participation in a
cross-linking reaction would otherwise likely interfere with
biological activity. When a cysteine residue is replaced, it is
typically desirable to minimize resulting changes in polypeptide
folding. Changes in polypeptide folding are minimized when the
replacement is chemically and sterically similar to cysteine. For
these reasons, serine is preferred as a replacement for cysteine.
As demonstrated in the examples below, a cysteine residue may be
introduced into a polypeptide's amino acid sequence for
cross-linking purposes. When a cysteine residue is introduced,
introduction at or near the amino or carboxy terminus is preferred.
Conventional methods are available for such amino acid sequence
modifications, whether the polypeptide of interest is produced by
chemical synthesis or expression of recombinant DNA.
[0099] Coupling of the two constituents can be accomplished via a
coupling or conjugating agent. There are several intermolecular
cross-linking reagents which can be utilized, See for example,
Means and Feeney, CHEMICAL MODIFICATION OF PROTEINS, Holden-Day,
1974, pp. 39-43. Among these reagents are, for example,
J-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) or
N,N'(1,3-phenylene) bismaleimide (both of which are highly specific
for sulfhydryl groups and form irreversible linkages);
N,N'-ethylene-bis-(iodoacetamide) or other such reagent having 6 to
11 carbon methylene bridges (which relatively specific for
sulfhydryl groups); and 1,5-difluoro-2,4-dinitrobenzene (which
forms irreversible linkages with amino and tyrosine groups). Other
crosslinking reagents useful for this purpose include:
p,p'-difluoro-m,m'-dinitrodiphenylsulfon- e (which forms
irreversible cross-linkages with amino and phenolic groups);
dimethyl adipimidate (which is specific for amino groups);
phenol-1,4-disulfonylchloride (which reacts principally with amino
groups); hexamethylenediisocyanate or diisothiocyanate, or
azophenyl-p-diisocyanate (which reacts principally with amino
groups); glutaraldehyde (which reacts with several different side
chains) and disdiazobenzidine (which reacts primarily with tyrosine
and histidine).
[0100] Cross-linking reagents may be homobifunctional, i.e., having
two functional groups that undergo the same reaction. A preferred
homobifunctional cross-linking reagent is bismaleimidohexane
("BMH"). BMH contains two maleimide functional groups, which react
specifically with sulfhydryl-containing compounds under mild
conditions (pH 6.5-7.7). The two maleimide groups are connected by
a hydrocarbon chain. Therefore, BMH is useful for irreversible
cross-linking of polypeptides that contain cysteine residues.
[0101] Cross-linking reagents may also be heterobifunctional.
Heterobifunctional cross-linking agents have two different
functional groups, for example an amine-reactive group and a
thiol-reactive group, that will cross-link two proteins having free
amines and thiols, respectively. Examples of heterobifunctional
cross-linking agents are succinimidyl 4-(N-maleimidomethyl)
cyclohexane-1-carboxylate ("SMCC"),
m-maleimidobenzoyl-N-hydroxysuccinimide ester ("MBS"), and
succinimide 4-(p-maleimidophenyl) butyrate ("SMPB"), an extended
chain analog of MBS. The succinimidyl group of these cross-linkers
reacts with a primary amine, and the thiolreactive maleimide forms
a covalent bond with the thiol of a cysteine residue.
[0102] Cross-linking reagents often have low solubility in water. A
hydrophilic moiety, such as a sulfonate group, may be added to the
cross-linking reagent to improve its water solubility. Sulfo-MBS
and sulfo-SMCC are examples of cross-linking reagents modified for
water solubility.
[0103] Many cross-linking reagents yield a conjugate that is
essentially non-cleavable under cellular conditions. However, some
cross-linking reagents contain a covalent bond, such as a
disulfide, that is cleavable under cellular conditions. For
example, Traut's reagent, dithiobis (succinimidylpropionate)
("DSP"), and N-succinimidyl 3-(2-pyridyldithio) propionate ("SPDP")
are well-known cleavable cross-linkers. The use of a cleavable
cross-linking reagent permits the cargo moiety to separate from the
transport polypeptide after delivery into the target cell. Direct
disulfide linkage may also be useful.
[0104] Numerous cross-linking reagents, including the ones
discussed above, are commercially available. Detailed instructions
for their use are readily available from the commercial suppliers.
A general reference on protein cross-linking and conjugate
preparation is: Wong, CHEMISTRY OF PROTEIN CONJUGATION AND
CROSS-LINKING, CRC Press (1991).
[0105] Chemical cross-linking may include the use of spacer arms.
Spacer arms provide intramolecular flexibility or adjust
intramolecular distances between conjugated moieties and thereby
may help preserve biological activity. A spacer arm may be in the
form of a polypeptide moiety that includes spacer amino acids, e.g.
proline. Alternatively, a spacer arm may be part of the
cross-linking reagent, such as in "long-chain SPDP" (Pierce Chem.
Co., Rockford, Ill., cat. No. 21651H).
[0106] Alternatively, the chimeric peptide can be produced as a
fusion peptide that includes the trafficking sequence and the JNK
inhibitor sequence which can conveniently be expressed in known
suitable host cells. Fusion peptides, as described herein, can be
formed and used in ways analogous to or readily adaptable from
standard recombinant DNA techniques, as describe above.
[0107] Production of Antibodies Specific for JNK Inhibitor
Peptides
[0108] JNK inhibitor peptides, including chimeric peptides
including the JNK inhibitor peptides (e.g., peptides including the
amino acid sequences shown in Table 1), as well peptides, or
derivatives, fragments, analogs or homologs thereof, may be
utilized as immunogens to generate antibodies that
immunospecifically-bind these peptide components. Such antibodies
include, e.g., polyclonal, monoclonal, chimeric, single chain, Fab
fragments and a Fab expression library. In a specific embodiment,
antibodies to human peptides are disclosed. In another specific
embodiment, fragments of the JNK inhibitor peptides are used as
immunogens for antibody production. Various procedures known within
the art may be used for the production of polyclonal or monoclonal
antibodies to a JNK inhibitor peptide, or derivative, fragment,
analog or homolog thereof.
[0109] For the production of polyclonal antibodies, various host
animals may be immunized by injection with the native peptide, or a
synthetic variant thereof, or a derivative of the foregoing.
Various adjuvants may be used to increase the immunological
response and include, but are not limited to, Freund's (complete
and incomplete), mineral gels (e.g., aluminum hydroxide), surface
active substances (e.g., lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, dinitrophenol, etc.) and human
adjuvants such as Bacille Calmette-Guerin and Corynebacterium
parvum.
[0110] For preparation of monoclonal antibodies directed towards a
JNK inhibitor peptide, or derivatives, fragments, analogs or
homologs thereof, any technique that provides for the production of
antibody molecules by continuous cell line culture may be utilized.
Such techniques include, but are not limited to, the hybridoma
technique (see, Kohler and Milstein, 1975. Nature 256: 495-497);
the trioma technique; the human B-cell hybridoma technique (see,
Kozbor, et al., 1983. Immunol Today 4: 72) and the EBV hybridoma
technique to produce human monoclonal antibodies (see, Cole, et
al., 1985. In: Monoclonal Antibodies and Cancer Therapy, Alan R.
Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized
in the practice of the present invention and may be produced by the
use of human hybridomas (see, Cote, et al., 1983. Proc Natl Acad
Sci USA 80: 2026-2030) or by transforming human B-cells with
Epstein Barr Virus in vitro (see, Cole, et al., 1985. In:
Monoclonal Antibodies and Cancer Therapy (Alan R. Liss, Inc., pp.
77-96).
[0111] According to the invention, techniques can be adapted for
the production of single-chain antibodies specific to a JNK
inhibitor peptide (see, e.g., U.S. Pat. No. 4,946,778). In
addition, methodologies can be adapted for the construction of Fab
expression libraries (see, e.g., Huse, et al., 1989. Science 246:
1275-1281) to allow rapid and effective identification of
monoclonal Fab fragments with the desired specificity for a JNK
inhibitor peptide or derivatives, fragments, analogs or homologs
thereof. Non-human antibodies can be "humanized" by techniques well
known in the art. See e.g., U.S. Pat. No. 5,225,539. Antibody
fragments that contain the idiotypes to a JNK inhibitor peptide may
be produced by techniques known in the art including, e.g., (i) an
F(ab').sub.2 fragment produced by pepsin digestion of an antibody
molecule; (ii) an Fab fragment generated by reducing the disulfide
bridges of an F(ab').sub.2 fragment; (iii) an Fab fragment
generated by the treatment of the antibody molecule with papain and
a reducing agent and (iv) Fv fragments.
[0112] In one embodiment, methodologies for the screening of
antibodies that possess the desired specificity include, but are
not limited to, enzyme-linked immunosorbent assay (ELISA) and other
immunologically-mediated techniques known within the art. In a
specific embodiment, selection of antibodies that are specific to a
particular domain of a JNK inhibitor peptide is facilitated by
generation of hybridomas that bind to the fragment of a JNK
inhibitor peptide possessing such a domain. Antibodies that are
specific for a domain within a JNK inhibitor peptide, or
derivative, fragments, analogs or homologs thereof, are also
provided herein.
[0113] The anti-JNK inhibitor peptide antibodies may be used in
methods known within the art relating to the localization and/or
quantitation of a JNK inhibitor peptide (e.g., for use in measuring
levels of the peptide within appropriate physiological samples, for
use in diagnostic methods, for use in imaging the peptide, and the
like). In a given embodiment, antibodies for the JNK inhibitor
peptides, or derivatives, fragments, analogs or homologs thereof
that contain the antibody derived binding domain, are utilized as
pharmacologically active compounds (hereinafter
"Therapeutics").
[0114] Methods of Treating or Preventing Disorders
[0115] Disorders Associated Undesired JNK Activity
[0116] Also included in the invention also are methods of treating
cell-proliferative disorders associated with JNK activation in a
subject by administering to a subject a biologically-active
therapeutic compound (hereinafter "Therapeutic").
[0117] The term "cell-proliferative disorder" denotes malignant as
well as non-malignant cell populations that often appear to differ
morphologically and functionally from the surrounding tissue. For
example, the method may be useful in treating malignancies of the
various organ systems, in which activation of JNK has often been
demonstrated, e.g. lung, breast, lymphoid, gastrointestinal, and
genito-urinary tract as well as adenocarcinomas which include
malignancies such as most colon cancers, renal-cell carcinoma,
prostate cancer, non-small cell carcinoma of the lung, cancer of
the small intestine and cancer of the esophagus. Cancers with
Bcr-Abl oncogenic transformations that clearly require activation
of JNK are also included.
[0118] The method is also useful in treating non-malignant or
immunological-related cell-proliferative diseases such as
psoriasis, pemphigus vulgaris, Behcet's syndrome, acute respiratory
distress syndrome (ARDS), ischemic heart disease, post-dialysis
syndrome, leukemia, rheumatoid arthritis, acquired immune
deficiency syndrome, vasculitis, septic shock and other types of
acute inflammation, and lipid histiocytosis. Especially preferred
are immunopathological disorders. Essentially, any disorder, which
is etiologically linked to JNK kinase activity, would be considered
susceptible to treatment.
[0119] Hearing Loss
[0120] Also included in the invention are methods of preventing or
treating hearing loss by administering to a subject a Therapeutic,
i.e., a cell-permeable bioactive peptide where the peptide prevents
damage to the hair cell stereocilia, hair cell apoptosis or
neuronal apoptosis. Preferably, the Therapeutic is the peptide of
SEQ ID NO:1, 2, 3, 4, 5, 6, 11, 12, 13, 14, 15, 16, 21, 22, 23, 24,
25, 26, 27 or 28.
[0121] Exposure to loud noise causes noise-induced hearing loss
(NIHL) by damaging the organs of the Corti, Damage an NIHL depends
upon both the level of the noise and the duration of the exposure.
Hearing loss may be temporary (TTS) if a repair mechanism is able
to restore the organ of the Corti, However it becomes permanent
(PTS) when hair cells or neurons die. Structural correlates to
noise trauma are of two types: (1) mild damage of synapses and or
hair cell stereocilia which can be repaired and accounts for TTS
and recovery and (2) severe damage inducing hair cell and neuronal
apoptosis which can not be repaired and accounts for PTS.
[0122] The Therapeutic is administered to the subject before
exposure to a noise trauma, antibiotic or chemotherapeutic agent.
Alternatively, the Therapeutic is administers after the subject is
exposed to a noise trauma, antibiotic or chemotherapeutic
agent.
[0123] A noise trauma is a noise which is sufficient to cause
damage to the corti. For example a noise trauma us at least 70 dB
SPL, at least 90 dB SPL or at least 100 dB SPL, at least 120 dB SPL
or at least 130 dB SPL.
[0124] Antibiotics include for example penicillins such as
penicillin G, penicillin V, ampicillin, amoxicillin, dicloxacillin,
and oxacillin; cephalosporins such as cephalexin (Keflex), cefaclor
(Ceclor), and cefixime (Suprax); aminoglycoside such as tobramycin,
and streptomycin; macrolides, such as erythromycin, azithromycin
(Zithromax) and clarithromycin; sulfonamides such as
trimethoprim-sulfamethoxazole or tetracylines such as tetracycline,
or doxycycline.
[0125] Neuronal Disorders
[0126] Also included in the invention are methods of treating or
preventing neuronal cell death related disorders by administering
to a cell or subject a composition of a cell-permeable bioactive
peptide where the peptide prevents damage to neurons or neuronal
apoptosis. The composition is for example the peptide of SEQ ID
NO:1, 2, 3, 4, 5, 6, 11, 12, 13, 14, 15, 16, or 21-26. Preferably,
the composition function to inhibit excitotoxic or oxidative
stress-induced death of neuronal cells. A neuronal cell is any cell
derived from the central or peripheral nervous system, e.g.,
neuron, neurite or dendrite. The cell is contacted in vivo, ex vivo
or in vitro. The subject is e.g., any mammal, e.g., a human, a
primate, mouse, rat, dog, cat, cow, horse, pig.
[0127] Neuronal cell death is measure by methods known in the art.
For example cell death is determined microscpopy or using a
chemical indicator such as Calcein-am (Molecular Probes).
[0128] Excitotoxicity is the main mechanism underlying neuronal
death in stroke, anoxic and traumatic brain damage is
excitotoxicity. Excitotoxicity is triggered by the excessive
activation of ionotropic glutamate-receptors, particularly, the
N-methyl-D-aspartate subtype of receptor, thereby leading to the
rapid influx of Ca.sup.2+ that triggers cell death. See e.g.,
Dirnagl et al., Trends in Neurosci. 22:391-97 (1999); Zipfel et
al., J of Neurotrauma 17:857-69 (2000).
[0129] The methods are useful to alleviate the symptoms of a
variety of neuronal disorders. The neuronal disorder is acute or
chronic. Neuronl disorder include those associated with n
excitotoxic event such as ischemic stroke, cerebral ischemia,
hypoxic/ischemic brain damage, traumatic brain damage, neuronal
death arising from epileptic seizures, and neuronal death
associated with several neurodegenerative disorders, such as
Alzheimer's disease, amyotrophic lateral sclerosis (ALS), and
Huntington's disease.
[0130] Neuronal death in cerebral ischemia is associated with
excitotoxic mechanisms. Cerebral ischemia (e.g., global cerebral
ischemia and focal cerebral ischemia) is due to stroke, head injury
or cardiac arrest. Global cerebral ischemia results from cardiac
arrest or bilateral carotid artery occlusion. Focal cerebral
ischemia results from a reduction in the cerebral blood flow
following cerebral artery occlusion. Focal cerebral ischemia
results in necrotic neuronal death by a complex pathogenic cascade
of events that includes energy depletion, excitotoxicity, and
peri-infarct depolarization, as well as a more delayed mechanism
involving both apoptosis and inflammation. Focal cerebral ischemia
can be further divided into thrombotic or embolic focal ischemia. A
thrombotic stroke occurs when cerebral arteries become blocked by a
blood clot that is formed within the brain. An embolic stroke is
also caused by a clotted artery, but in embolic stroke, the clot is
formed somewhere other than in the brain itself.
[0131] The methods described herein lead to a reduction in the
severity or the alleviation of one or more symptoms of an neuronal
disorder such as those described herein. Neuronal disorders are
diagnosed and or monitored, typically by a physician using standard
methodologies.
[0132] The JNKI peptides of the invention have been shown, as
described in the Examples, to provide high levels of
neuroprotection, and moreover, the level of protection remains high
even when the JNKI peptide is administered 6-12 hours after the
onset of ischemia. While 30-50% neuroprotection has been
demonstrated with various compounds administered up to 9 hours
postischemia (see e.g., Fink et al., J. Cereb. Blood Flow Metab.,
18:1071-76 (1998); Williams et al., Neuroreport, 13:821-24 (2002),
each of which is hereby incorporated by reference in its entirety),
the JNKI peptides of the present invention have been shown to
provide protection when administered 12 hours after the ischemic
event, as described in the Examples below.
[0133] Most patients that are experiencing, or have experienced, an
excitotoxic event, such as an ischemic stroke, seek medical
assistance within 3 to 6 hours following the excitotoxic event. The
length of time available to treat or prevent excitotoxic damage,
such as the damage that occurs in an ischemic event, is important,
as activation of the cell death machinery can take several hours
following the excitotoxic event. Accordingly, the Therapeutic can
be administered to the subject before experiencing an excitotoxic
event. Alternatively, the Therapeutic can be administered after the
subject experiences an excitotoxic event.
[0134] Inhibiting Pancreatic Islet Cell Death
[0135] Also include are methods of inhibiting cell damage or death
or preventing aberrant cell damage such as oxidative-stress induced
cell death (e.g., apoptotic cell death) by administering to a
subject a bioactive therapeutic administering to a cell or subject
a composition (Therapeutic) containing a bioactive peptide where
the peptide prevents cell death or damage. The cell is for example
a pancreatic cell. Preferably, the Therapeutic is the peptide of
SEQ ID NO:1, 2, 3, 4, 5, 6, 11, 12, 13, 14, 15, 16, or 21-26.
Optionally, the cell or subject is also administered
collagenase.
[0136] The peptide is administered either before or after the
subject is exposed to a proinflammatory cytokine such as the
interleukins.
[0137] The subject can be e.g., any mammal, e.g., a human, a
primate, mouse, rat, dog, cat, cow, horse, pig.
[0138] Pharmaceutical Compositions
[0139] The Therapeutics include, e.g.: (i) any one or more of the
JNK inhibitor peptides, and derivative, fragments, analogs and
homologs thereof; (ii) antibodies directed against the JNK
inhibitor peptides; (iii) nucleic acids encoding a JNK inhibitor
peptide, and derivatives, fragments, analogs and homologs thereof;
(iv) antisense nucleic acids to sequences encoding a JNK inhibitor
peptide, and (v) modulators (i.e., inhibitors, agonists and
antagonists).
[0140] The term "therapeutically effective" means that the amount
of inhibitor peptide, for example, which is used, is of sufficient
quantity to ameliorate the JNK associated disorder.
[0141] Treatment includes administration of a reagent that
modulates JNK kinase activity. The term "modulate" includes the
suppression of expression of JNK when it is over-expressed. It also
includes suppression of phosphorylation of c jun, ATF2 or NFAT4,
for example, by using a peptide of any one or more of SEQ ID NOs:
1-6, and 21-22 and SEQ ID NOs: 11-16 and 23-26 as a competitive
inhibitor of the natural c-jun ATF2 and NFAT4 binding site in a
cell. Thus also includes suppression of hetero- and homo-meric
complexes of transcription factors made up of cjun, ATF2, or NFAT4
and their related partners, such as for example the AP-1 complex
that is made up of c-jun, AFT2 and c-fos. When a cell proliferative
disorder is associated with JNK overexpression, such suppressive
JNK inhibitor peptides can be introduced to a cell. In some
instances, "modulate" may include the increase of JNK expression,
for example by use of an IB peptide-specific antibody that blocks
the binding of an IB-peptide to JNK, thus preventing JNK inhibition
by the IB-related peptide. The JNK inhibitor, peptides, fusion
peptides and nucleic acids of the invention can be formulated in
pharmaceutical compositions. These compositions may comprise, in
addition to one of the above substances, a pharmaceutically
acceptable excipient, carrier, buffer, stabiliser or other
materials well known to those skilled in the art. Such materials
should be non-toxic and should not interfere with the efficacy of
the active ingredient. The precise nature of the carrier or other
material may depend on the route of administration, e.g. oral,
intravenous, cutaneous or subcutaneous, nasal, intramuscular,
intraperitoneal, intrauricular, or patch routes.
[0142] Pharmaceutical compositions for oral administration may be
in tablet, capsule, powder or liquid form. A tablet may include a
solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical
compositions generally include a liquid carrier such as water,
petroleum, animal or vegetable oils, mineral oil or synthetic oil.
Physiological saline solution, dextrose or other saccharide
solution or glycols such as ethylene glycol, propylene glycol or
polyethylene glycol may be included.
[0143] For intravenous, cutaneous or subcutaneous injection, or
injection at the site of affliction, the active ingredient will be
in the form of a parenterally acceptable aqueous solution which is
pyrogen-free and has suitable pH, isotonicity and stability. Those
of relevant skill in the art are well able to prepare suitable
solutions using, for example, isotonic vehicles such as Sodium
Chloride Injection, Ringer's Injection, Lactated Ringer's
Injection. Preservatives, stabilisers, buffers, antioxidants and/or
other additives may be included, as required.
[0144] Whether it is a polypeptide, peptide, or nucleic acid
molecule, other pharmaceutically useful compound according to the
present invention that is to be given to an individual,
administration is preferably in a "prophylactically effective
amount" or a "therapeutically effective amount" (as the case may
be, although prophylaxis may be considered therapy), this being
sufficient to show benefit to the individual. The actual amount
administered, and rate and time-course of administration, will
depend on the nature and severity of what is being treated.
Prescription of treatment, e.g. decisions on dosage etc, is within
the responsibility of general practitioners and other medical
doctors, and typically takes account of the disorder to be treated,
the condition of the individual patient, the site of delivery, the
method of administration and other factors known to practitioners.
Examples of the techniques and protocols mentioned above can be
found in REMINGTON'S PHARMACEUTICAL SCIENCES, 16th edition, Osol,
A. (ed), 1980.
[0145] Alternatively, targeting therapies may be used to deliver
the active agent more specifically to certain types of cell, by the
use of targeting systems such as antibody or cell specific ligands.
Targeting may be desirable for a variety of reasons; for example if
the agent is unacceptably toxic, or if it would otherwise require
too high a dosage, or if it would not otherwise be able to enter
the target cells.
[0146] Instead of administering these agents directly, they could
be produced in the target cells by expression from an encoding gene
introduced into the cells, e.g. in a viral vector (a variant of the
VDEPT technique--see below). The vector could be targeted to the
specific cells to be treated, or it could contain regulatory
elements, which are switched on more or less selectively by the
target cells.
[0147] Alternatively, the agent could be administered in a
precursor form, for conversion to the active form by an activating
agent produced in, or targeted to, the cells to be treated. This
type of approach is sometimes known as ADEPT or VDEPT; the former
involving targeting the activating agent to the cells by
conjugation to a cell-specific antibody, while the latter involves
producing the activating agent, e.g. a JNK inhibitor peptide, in a
vector by expression from encoding DNA in a viral vector (see for
example, EP-A-415731 and WO 90/07936).
[0148] In a specific embodiment of the present invention, nucleic
acids include a sequence that encodes a JNK inhibitor peptide, or
functional derivatives thereof, are administered to modulate
activated JNK signaling pathways by way of gene therapy. In more
specific embodiments, a nucleic acid or nucleic acids encoding a
JNK inhibitor peptide, or functional derivatives thereof, are
administered by way of gene therapy. Gene therapy refers to therapy
that is performed by the administration of a specific nucleic acid
to a subject. In this embodiment of the present invention, the
nucleic acid produces its encoded peptide(s), which then serve to
exert a therapeutic effect by modulating function of the disease or
disorder. Any of the methodologies relating to gene therapy
available within the art may be used in the practice of the present
invention. See e.g., Goldspiel, et al., 1993. Clin Pharm 12:
488-505.
[0149] In a preferred embodiment, the Therapeutic comprises a
nucleic acid that is part of an expression vector expressing any
one or more of the IB-related peptides, the JBD.sub.20-related
peptides or fragments, derivatives or analogs thereof, within a
suitable host. In a specific embodiment, such a nucleic acid
possesses a promoter that is operably-linked to coding region(s) of
a JNK inhibitor peptide. The promoter may be inducible or
constitutive, and, optionally, tissue-specific. In another specific
embodiment, a nucleic acid molecule is used in which coding
sequences (and any other desired sequences) are flanked by regions
that promote homologous recombination at a desired site within the
genome, thus providing for intra-chromosomal expression of nucleic
acids. See e.g., Koller and Smithies, 1989. Proc Natl Acad Sci USA
86: 8932-8935.
[0150] Delivery of the Therapeutic nucleic acid into a patient may
be either direct (i.e., the patient is directly exposed to the
nucleic acid or nucleic acid-containing vector) or indirect (i.e.,
cells are first transformed with the nucleic acid in vitro, then
transplanted into the patient). These two approaches are known,
respectively, as in vivo or ex vivo gene therapy. In a specific
embodiment of the present invention, a nucleic acid is directly
administered in vivo, where it is expressed to produce the encoded
product. This may be accomplished by any of numerous methods known
in the art including, e.g., constructing the nucleic acid as part
of an appropriate nucleic acid expression vector and administering
the same in a manner such that it becomes intracellular (e.g., by
infection using a defective or attenuated retroviral or other viral
vector; see U.S. Pat. No. 4,980,286); directly injecting naked DNA;
using microparticle bombardment (e.g., a "Gene Gun.RTM.; Biolistic,
DuPont); coating the nucleic acids with lipids; using associated
cell-surface receptors/transfecting agents; encapsulating in
liposomes, microparticles, or microcapsules; administering it in
linkage to a peptide that is known to enter the nucleus; or by
administering it in linkage to a ligand predisposed to
receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987. J Biol
Chem 262: 4429-4432), which can be used to "target" cell types that
specifically express the receptors of interest, etc.
[0151] An additional approach to gene therapy in the practice of
the present invention involves transferring a gene into cells in in
vitro tissue culture by such methods as electroporation,
lipofection, calcium phosphate-mediated transfection, viral
infection, or the like. Generally, the method of transfer includes
the concomitant transfer of a selectable marker to the cells. The
cells are then placed under selection pressure (e.g., antibiotic
resistance) so as to facilitate the isolation of those cells that
have taken up, and are expressing, the transferred gene. Those
cells are then delivered to a patient. In a specific embodiment,
prior to the in vivo administration of the resulting recombinant
cell, the nucleic acid is introduced into a cell by any method
known within the art including, e.g., transfection,
electroporation, microinjection, infection with a viral or
bacteriophage vector containing the nucleic acid sequences of
interest, cell fusion, chromosome-mediated gene transfer,
microcell-mediated gene transfer, spheroplast fusion, and similar
methodologies that ensure that the necessary developmental and
physiological functions of the recipient cells are not disrupted by
the transfer. See e.g., Loeffler and Behr, 1993. Meth Enzymol 217:
599-618. The chosen technique should provide for the stable
transfer of the nucleic acid to the cell, such that the nucleic
acid is expressible by the cell. Preferably, the transferred
nucleic acid is heritable and expressible by the cell progeny.
[0152] In preferred embodiments of the present invention, the
resulting recombinant cells may be delivered to a patient by
various methods known within the art including, e.g., injection of
epithelial cells (e.g., subcutaneously), application of recombinant
skin cells as a skin graft onto the patient, and intravenous
injection of recombinant blood cells (e.g., hematopoietic stem or
progenitor cells). The total amount of cells that are envisioned
for use depend upon the desired effect, patient state, and the
like, and may be determined by one skilled within the art.
[0153] Cells into which a nucleic acid can be introduced for
purposes of gene therapy encompass any desired, available cell
type, and may be xenogeneic, heterogeneic, syngeneic, or
autogeneic. Cell types include, but are not limited to,
differentiated cells such as epithelial cells, endothelial cells,
keratinocytes, fibroblasts, muscle cells, hepatocytes and blood
cells, or various stem or progenitor cells, in particular embryonic
heart muscle cells, liver stem cells (International Patent
Publication WO 94/08598), neural stem cells (Stemple and Anderson,
1992, Cell 71: 973-985), hematopoietic stem or progenitor cells,
e.g., as obtained from bone marrow, umbilical cord blood,
peripheral blood, fetal liver, and the like. In a preferred
embodiment, the cells utilized for gene therapy are autologous to
the patient.
[0154] Immunoassays
[0155] The peptides and antibodies of the present invention may be
utilized in assays (e.g., immunoassays) to detect, prognose,
diagnose, or monitor various conditions, diseases, and disorders
characterized by aberrant levels of JNK, or a JNK inhibitor
peptide, or monitor the treatment thereof. An "aberrant level"
means an increased or decreased level in a sample relative to that
present in an analogous sample from an unaffected part of the body,
or from a subject not having the disorder. The immunoassay may be
performed by a method comprising contacting a sample derived from a
patient with an antibody under conditions such that
immunospecific-binding may occur, and subsequently detecting or
measuring the amount of any immunospecific-binding by the antibody.
In a specific embodiment, an antibody specific for a JNK inhibitor
peptide may be used to analyze a tissue or serum sample from a
patient for the presence of JNK or a JNK inhibitor peptide; wherein
an aberrant level of JNK or a JNK inhibitor peptide is indicative
of a diseased condition. The immunoassays that may be utilized
include, but are not limited to, competitive and non-competitive
assay systems using techniques such as Western Blots,
radioimmunoassays (RIA), enzyme linked immunosorbent assay (ELISA),
"sandwich" immunoassays, immunoprecipitation assays, precipitin
reactions, gel diffusion precipitin reactions, immunodiffusion
assays, agglutination assays, fluorescent immunoassays,
complement-fixation assays, immunoradiometric assays, and protein-A
immunoassays, etc.
[0156] Kits
[0157] The present invention additionally provides kits for
diagnostic or therapeutic use that include one or more containers
containing an anti-JNK inhibitor peptide antibody and, optionally,
a labeled binding partner to the antibody. The label incorporated
into the antibody may include, but is not limited to, a
chemiluminescent, enzymatic, fluorescent, calorimetric or
radioactive moiety. In another specific embodiment, kits for
diagnostic use that are comprised of one or more containers
containing modified or unmodified nucleic acids that encode, or
alternatively, that are the complement to, a JNK inhibitor peptide
and, optionally, a labeled binding partner to the nucleic acids,
are also provided. In an alternative specific embodiment, the kit
may comprise, in one or more containers, a pair of oligonucleotide
primers (e.g., each 6-30 nucleotides in length) that are capable of
acting as amplification primers for polymerase chain reaction (PCR;
see, e.g., Innis, et al., 1990. PCR PROTOCOLS, Academic Press,
Inc., San Diego, Calif.), ligase chain reaction, cyclic probe
reaction, and the like, or other methods known within the art. The
kit may, optionally, further comprise a predetermined amount of a
purified JNK inhibitor peptide, or nucleic acids thereof, for use
as a diagnostic, standard, or control in the assays.
[0158] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and accompanying figures. Such modifications
fall within the scope of the appended claims.
[0159] Various publications are cited herein, the disclosures of
which are incorporated by reference in their entirety.
SPECIFIC EXAMPLES
Example 1
Identification of JNK Inhibitor Peptides
[0160] Amino acid sequences important for efficient interaction
with JNK were identified by sequence alignments between known JBDs.
Sequence comparison between the JBDs of IB1 [SEQ ID NO:17], IB2
[SEQ ID NO:18], c-Jun [SEQ ID NO:19] and ATF2 [SEQ ID NO:20]
defined a weakly conserved 8 amino acid sequence (FIG. 1A). Since
the JBDs of IB1 and IB2 are approximately 100 fold as efficient as
c-Jun or ATF2 in binding JNK (Dickens et al. Science 277: 693
(1997), it was reasoned that conserved residues between IB1 and IB2
must be important to confer maximal binding. The comparison between
the JBDs of IB1 and IB2 defined two blocks of seven and three amino
acids that are highly conserved between the two sequences. These
two blocks are contained within a peptide sequence of 23 amino
acids in IB1 [SEQ ID NO:1] and 21 amino acid IB2 [SEQ ID NO:2].
These sequences are shown in FIG. 1B, dashes in the IB2 sequence
indicate a gap in the sequence in order to align the conserved
residues.
[0161] The JNK inhibitor (JNKI) peptides of the present invention
were obtained by linking the 20 amino acid JNK-binding motif of
JIP-1/IB 1, referred to herein as JBD.sub.20, to a trafficking
protein, such as for example, the 10 amino acid HIV-TAT.sub.48-57
transporter sequence.
Example 2
Preparation of JNK Inhibitor Fusion Proteins
[0162] JNK inhibitor fusion proteins were synthesized by covalently
linking the C-terminal end of JBD.sub.23 or the 21 amino acid
sequence derived from the JBD of IB2 (JBD.sub.21) or the C-terminal
end of the JBD.sub.20 amino acid sequence to a N-terminal 10 amino
acid long carrier peptide derived from the HIV-TAT.sub.48-57 (Vives
et al., J. Biol. Chem. 272: 16010 (i 997)) via a spacer consisting
of two proline residues. This spacer was used to allow for maximal
flexibility and prevent unwanted secondary structural changes. As
shown in FIG. 1C, these preparations were designated L-TAT [SEQ ID
NO:7], L-TAT-IB 1 [SEQ ID NO: 11],L-TAT-IB2 [SEQ ID NO: 12] and
L-TAT-JNKI1 [SEQ ID NO:21], respectively. All-D retro-inverso
peptides TAT-fusion peptides were also synthesized and were
designated D-TAT [SEQ ID NO:8], D-TAT-IB1 [SEQ ID NO:14], AND
DTAT-JNKI1 [SEQ ID NO:22] respectively. All D and L peptides were
produced by classical F-mock synthesis and further analysed by Mass
Spectrometry. They were finally purified by HPLC. To determine the
effects of the proline spacer, two types of TAT peptide were
produced one with and one without two prolines. The addition of the
two prolines did not appear to modify the entry or the localization
of the TAT peptide inside cells.
[0163] Generic peptides showing the conserved amino acid residues
are given in FIG. 2. An "X" indicates any amino acid. The number of
Xs in a given peptide is not limited to the one depicted, and may
vary (i.e., X can represent any number of amino acid residues,
including zero). See above for a more detailed description of the
generic sequences.
Example 3
Inhibition of .beta.Cell Death By JBD.sub.23
[0164] Effects of the 23 a.a. long JBD sequence of IB1 on JNK
biological activities were then studied. The 23 a.a. sequence was
linked N-terminal to the Green Fluorescent Protein (GFP-JBD.sub.23
construct), and the effect of this construct on pancreatic
.beta.-cell apoptosis induced by IL-1.beta. was evaluated. See FIG.
3. This mode of apoptosis was previously shown to be blocked by
transfection with JBD.sub.1-280, whereas specific inhibitors of
ERK1/2 or p38 did not protect. See Ammendrup et al, supra.
[0165] Oligonucleotides corresponding to the 23 amino acid sequence
(JBD.sub.23; FIG. 1B) and a sequence mutated at the fully conserved
regions (JBD.sub.23mut) were synthesized and directionally inserted
into the EcoRI and SalI sites of the pEGFP-N1 vector encoding the
Green Fluorescent Protein (GFP) (from Clontech). Insulin producing
.beta.TC-3 cells were cultured in RPMI 1640 medium supplemented
with 10% Fetal Calf Serum, 100 .mu.g/mL Streptomycin, 100 units/mL
Penicillin and 2 mM Glutamine. Insulin producing .beta.TC-3 cells
were transfected with the indicated vectors and IL-1.beta. (10
ng/mL) was added to the cell culture medium. The number of
apoptotic cells were counted at 48 hours after the addition of
IL-1.beta. using an inverted fluorescence microscope. Apoptotic
cells were discriminated from normal cells by the characteristic
"blebbing out" of the cytoplasm were counted after two days.
[0166] As indicated in FIG. 3, GFP is Green Fluorescent protein
expression vector used as a control; JBD23 is the vector expressing
a chimeric GFP linked to the 23 a.a. sequence from the JBD of IB1;
JBD23Mut is the same vector as GFP-JBD23, but with a JBD mutated at
four conserved residues shown as FIG. 1B; and JBD280 is the GFP
vector linked to the entire JBD (a.a. 1-280). The GFP-JBD.sub.23
expressing construct prevented IL-1.beta. induced pancreatic
.beta.-cell apoptosis as efficiently as the entire JBD.sub.1-280
(FIG. 3, JBD23/IL-1 compared to JBD280/IL-1). As additional
controls, sequences mutated at fully conserved IB1 residues had
greatly decreased ability to prevent apoptosis (FIG. 3,
JBD23Mut/IL-1).
Example 4
Cellular Import Of TAT-IB1 and TAT-IB2 Peptides
[0167] The ability of the L- and D-enantiomeric forms of TAT,
TAT-IB1 and TAT-IB2 peptides ("TAT-IB peptides") to enter cells
were evaluated.
[0168] L-TAT, D-TAT, L-TAT-IB1, L-TAT-IB2 and D-TAT-IB1 peptides
[SEQ ID NOs:7, 8, 11, 12 and 14, respectively] were labeled by
N-terminal addition of a glycine residue conjugated to fluorescein.
Labeled peptides (1 .mu.M) were added to .beta.TC-3 cell cultures,
which were maintained as described in Example 3. At predetermined
times, cells were washed with PBS and fixed for five minutes in
ice-cold methanol-acetone (1:1) before being examined under a
fluorescence microscope. Fluorescein-labeled BSA (1 .mu.M, 12
moles/mole BSA) was used as a control. Results demonstrated that
all the above fluorescein labeled peptides had efficiently and
rapidly (less than five minutes) entered cells once added to the
culture medium. Conversely, fluorescein labeled bovine serum
albumin (1 .mu.M BSA, 12 moles fluorescein/mole BSA) did not enter
the cells.
[0169] A time course study indicated that the intensity of the
fluorescent signal for the L-enantiomeric peptides decreased by 70%
following a 24 hours period. Little to no signal was present at 48
hours. In contrast, D-TAT and D-TAT-IB1 were extremely stable
inside the cells. Fluorescent signals from these all-D
retro-inverso peptides were still very strong 1 week later, and the
signal was only slightly diminish at 2 weeks post treatment.
Example 5
In Vitro Inhibition Of c-JUN, ATF2 and Elk1 Phosphorylation
[0170] The effects of the peptides on JNKs-mediate phosphorylation
of their target transcription factors were investigated in vitro.
Recombinant and nonactivated JNK1, JNK2 and JNK3 were produced
using a TRANSCRIPTION AND TRANSLATION rabbit reticulocyte lysate
kit (Promega) and used in solid phase kinase assays with c-Jun,
ATF2 and Elk1, either alone or fused to glutathione-S-transferase
(GST), as substrates. Dose response studies were performed wherein
L-TAT, L-TAT-IB1 or L-TAT-IB2 peptides (0-25 .mu.M) were mixed with
the recombinant JNK1, JNK2, or JNK3 kinases in reaction buffer (20
mM Tris-acetate, 1 mM EGTA, 10 mM p-nitrophenyl-phosphate (pNPP), 5
mM sodium pyrophosphate, 10 mM p-glycerophosphate, 1 mM
dithiothreitol) for 20 minutes. The kinase reactions were then
initiated by the addition of 10 mM MgCl.sub.2 and 5 .mu.Ci
.sup.33P-.gamma.-dATP and 1 pg of either GST-Jun (a.a. 1-89),
GST-AFT2 (a.a. 1-96) or GST-ELK1 (a.a. 307-428). GST-fusion
proteins were purchased from Stratagene (La Jolla, Calif.). Ten
.mu.L of glutathione-agarose beads were also added to the mixture.
Reaction products were then separated by SDS-PAGE on a denaturing
10% polyacrylamide gel. Gels were dried and subsequently exposed to
X-ray films (Kodak). Nearly complete inhibition of c-Jun, ATF2 and
Elk1 phosphorylation by JNKs was observed at TAT-IB peptide doses
as low as 2.5 .mu.M. However, a marked exception was the absence of
TAT-IB inhibition of JNK3 phosphorylation of Elk1. Overall, the
TAT-IB1 peptide appeared slightly superior to TAT-IB2 in inhibiting
JNK family phosphorylation of their target transcription factors.
(See, FIG. 4A).
[0171] The ability of D-TAT, D-TAT-IB1 and L-TAT-IB1 peptides
(0-250 .mu.M dosage study) to inhibit GST-Jun (a.a. 1-73)
phosphorylation by recombinant JNK1, JNK2, and JNK3 by were
analyzed as described above. Overall, D-TAT-IB1 peptide decreased
JNK-mediated phosphorylation of c-Jun, but at levels approximately
10-20 fold less efficiently than L-TAT-IB1. (See, FIG. 4B).
Example 6
Inhibition of c-JUN Phosphorylation By Activated JNKs
[0172] The effects of the L-TAT, L-TAT-IB1 or L-TAT-IB2 peptides on
JNKs activated by stressful stimuli were evaluated using GST-Jun to
pull down JNKs from UV-light irradiated HeLa cells or IL-1.beta.
treated .beta.TC cells. .beta.TC cells were cultured as described
above. HeLa cells were cultured in DMEM medium supplemented with
10% Fetal Calf Serum, 100 .mu.g/mL Streptomycin, 100 units/ml
Penicillin and 2 mM Glutamine. One hour prior to being used for
cell extract preparation, .beta.TC cells were activated with
IL-1.beta. as described above, whereas HeLa cells were activated by
UV-light (20 J/m.sup.2). Cell extracts were prepared from control,
UV-light irradiated HeLa cells and IL-1.beta. treated .beta.TC-3
cells by scraping the cell cultures in lysis buffer (20 mM
Tris-acetate, 1 mM EGTA, 1% Triton X-100, 10 mM
p-nitrophenyl-phosphate, 5 mM sodium pyrophosphate, 10 mM
.beta.-glycerophosphate, 1 mM dithiothretiol). Debris was removed
by centrifugation for five minutes at 15,000 rpm in an SS-34
Beckman rotor. One-hundred .mu.g extracts were incubated for one
hour at room temperature with one .mu.g GST-jun (amino acids 1-89)
and 10 .mu.L of glutathione-agarose beads (Sigma). Following four
washes with the scraping buffer, the beads were resuspended in the
same buffer supplemented with L-TAT, L-TAT-IB1 or L-TAT-IB2
peptides (25 .mu.M) for 20 minutes. Kinase reactions were then
initiated by the addition of 10 mM MgCl.sub.2 and 5 .mu.Ci
.sup.33P-.gamma.-dATP and incubated for 30 minutes at 30.degree. C.
Reaction products were then separated by SDS-PAGE on a denaturing
10% polyacrylamide gel. Gels were dried and subsequently exposed to
X-ray films (Kodak). The TAT-IB peptides efficiently prevented
phosphorylation of c-Jun by activated JNKs in these experiments.
(See, FIG. 6).
Example 7
In Vivo Inhibition of c-JUN Phosphorylation By TAT-IB Peptides
[0173] To determine whether the cell-permeable peptides could block
JNK signaling in vivo, we used a heterologous GAL4 system. HeLa
cells, cultured as described above, were co-transfected with the
5xGAL-LUC reporter vector together with the GAL-Jun expression
construct (Stratagene) comprising the activation domain of c-Jun
(amino acids 1-89) linked to the GAL4 DNA-binding domain.
Activation of JNK was achieved by the co-transfection of vectors
expressing the directly upstream kinases MKK4 and MKK7 (See,
Whitmarsh et al., Science 285: 1573 (1999)). Briefly,
3.times.10.sup.5 cells were transfected with the plasmids in 3.5-cm
dishes using DOTAP (Boehringer Mannheim) following instructions
from the manufacture. For experiments involving GAL-Jun, 20 ng of
the plasmid was transfected with 1 .mu.g of the reporter plasmid
pFR-Luc (Stratagene) and 0.5 .mu.g of either MKK4 or MKK7
expressing plasmids. Three hours following transfection, cell media
were changed and TAT, TAT-IB 1, and TAT-IB2 peptides (1 .mu.M) were
added. The luciferase activities were measured 16 hours later using
the "Dual Reporter System" from Promega after normalization to
protein content. As shown in FIG. 5, addition of both the TAT-IB1
and TAT-IB2 peptides blocked activation of c-Jun following MKK4 and
MKK7 mediated activation of JNK. Because HeLa cells express both
JNKI and JNK2 isoforms but not JNK3, we transfected cells with
JNK3. Again, the two TAT-IB peptides inhibited JNK2 mediated
activation of c-Jun.
Example 8
Inhibition of IL-1.beta. Induced Pancreatic .beta.-Cell Death By
TAT-IB Peptides
[0174] We investigated the effects of the L-TAT-IB peptides on the
promotion of pancreatic .beta.-cell apoptosis elicited by
IL-1.beta.. .beta.TC-3 cell cultures were incubated for 30 minutes
with 1 .mu.M of either L-TAT-IB1 or L-TAT-IB2 peptides followed by
10 ng/mL of IL-1.beta.. A second addition of peptide (1 .mu.M) was
performed 24 hours later. Apoptotic cells were counted after two
days of incubation with IL-1.beta. using Propidium Iodide (red
stained cell are dead cells) and Hoechst 33342 (blue stained cell
are cells with intact plasma membrane) nuclear staining. As shown
in FIG. 5, addition of the TAT-IB peptides inhibited
IL-1.beta.-induced apoptosis of .beta.TC-3 cells cultured in the
presence of IL-1.beta. for two days.
[0175] Long term inhibition of IL-1.beta. induced cells death was
examined by treating .beta.TC-3 cells as described above, except
that incubation of the cells with the peptides and IL-1.beta. was
sustained for 12 days. Additional peptides (1 .mu.M) were added
each day and additional IL-1.beta. (10 ng/mL) was added every 2
days. The TAT-IB1 peptide confers strong protection against
apoptosis in these conditions. Taken together, these experiments
establish that TAT-IB peptides are biologically active molecules
able to prevent the effects of JNK signaling on cell fate.
Example 9
Synthesis of an All-D-Retro-Inverso Peptides
[0176] Peptides of the invention may be all-D amino acid peptides
synthesized in reverse to prevent natural proteolysis (i.e.,
all-D-retro-inverso peptides). An all-D retro-inverso peptide of
the invention would provide a peptide with functional properties
similar to the native peptide, wherein the side groups of the
component amino acids would correspond to the native peptide
alignment, but would retain a protease resistant backbone.
[0177] Retro-inverso peptides of the invention are analogs
synthesized using D-amino acids by attaching the amino acids in a
peptide chain such that the sequence of amino acids in the
retro-inverso peptide analog is exactly opposite of that in the
selected peptide which serves as the model. To illustrate, if the
naturally occurring TAT protein (formed of L-amino acids) has the
sequence GRKKRRQRRR [SEQ ID NO:7], the retro-inverso peptide analog
of this peptide (formed of D-amino acids) would have the sequence
RRRQRRKKRG [SEQ ID NO:8]. The procedures for synthesizing a chain
of D-amino acids to form the retro-inverso peptides are known in
the art. See, e.g., Jameson et al., Nature, 368, 744-746 (1994);
Brady et al., Nature, 368, 692-693 (1994)); Guichard et al., J.
Med. Chem. 39, 2030-2039 (1996). Specifically, the retro-peptides
were produced by classical F-mock synthesis and further analysed by
Mass Spectrometry. They were finally purified by HPLC.
[0178] Since an inherent problem with native peptides is
degradation by natural proteases and inherent immunogenicity, the
heterobivalent or heteromultivalent compounds of this invention
will be prepared to include the "retro-inverso isomer" of the
desired peptide. Protecting the peptide from natural proteolysis
should therefore increase the effectiveness of the specific
heterobivalent or heteromultivalent compound, both by prolonging
half-life and decreasing the extent of the immune response aimed at
actively destroying the peptides.
Example 10
Long Term Biological Activity of all-D-Retro-Inverso IB
Peptides
[0179] Long term biological activity is predicted for the D-TAT-IB
retro-inverso containing peptide heteroconjugate when compared to
the native L-amino acid analog owing to protection of the D-TAT-IB
peptide from degradation by native proteases, as shown in Example
5.
[0180] Inhibition of IL-1.beta. induced pancreatic P-cell death by
the D-TAT-IB1 peptide was analyzed. As shown in FIG. 10, .beta.TC-3
cells were incubated as described above for 30 minutes with one
single addition of the indicated peptides (1 .mu.M), then
IL-1.beta. (10 ng/ml) was added. Apoptotic cells were then counted
after two days of incubation with IL-1.beta. by use of Propidium
Iodide and Hoechst 33342 nuclear staining. A minimum of 1,000 cells
were counted for each experiment. Standard Error of the Means (SEM)
are indicated, n=5. The D-TAT-IB1 peptide decreased IL-1 induced
apoptosis to a similar extent as L-TAT-IB peptides (compare FIG. 5
and FIG. 10).
[0181] Long term inhibition of IL-1.beta. induced cell-death by the
D-TAT-IB1 peptide was also analyzed. .beta.TC-3 cells were
incubated as above for 30 minutes with one single addition of the
indicated peptides (1 .mu.M), then IL-1.beta. (10 ng/ml) was added,
followed by addition of the cytokine every two days. Apoptotic
cells were then counted after 15 days of incubation with IL-1.beta.
by use of Propidium Iodide and Hoechst 33342 nuclear staining. Note
that one single addition of the L-TAT-IB1 peptide does not confer
long-term protection. A minimum of 1,000 cells were counted for
each experiment. Standard Error of the Means (SEM) are indicated,
n=5. Results are shown in FIG. 9. D-TAT-IB 1, but not L-TAT-IB 1,
was able to confer long term (15 day) protection.
Example 11
Inhibition of Irradiation Induced Pancreatic p-Cell Death By TAT-IB
Peptides
[0182] JNK is also activated by ionizing radiation. To determine
whether TAT-IB peptides would provide protection against
radiation-induced JNK damage, "WiDr" cells were irradiated (30 Gy)
in presence or absence of D-TAT, L-TAT-IB 1 or D-TAT-IB 1 peptides
(1 .mu.M added 30 minutes before irradiation), as indicated in FIG.
10. Control cells (CTRL) were not irradiated. Cells were analyzed
48 hours later by mean of PI and Hoechst 33342 staining, as
described above. n=3, SEM are indicated. L-TAT-IB1 and D-TAT-IB1
peptides were both able to prevent irradiation induced apoptosis in
this human colon cancer cell line.
Example 12
Radioprotection to Ionizing Radiation By TAT-IB Peptides
[0183] To determine the radioprotective effects of the TAT-IB
peptides, C57 B1/6 mice (2 to 3 months old) were irradiated with a
Phillips RT 250 R-ray at a dose rate of 0.74 Gy/min (17 mA, 0.5 mm
Cu filter). Thirty minutes prior to irradiation, the animals were
injected i.p. with either the TAT, L-TAT-IB1 and D-TAT-IB1 peptides
(30 .mu.l of a 1 mM solution). Briefly, mice were irradiated as
follows: mice were placed in small plastic boxes with the head
lying outside the box. The animals were placed on their back under
the irradiator, and their neck fixed in a small plastic tunnel to
maintain their head in a correct position. The body was protected
with lead. Prior to irradiation mice were maintained on standard
pellet mouse chow, however post irradiation mice were fed with a
semi-liquid food that was renewed each day.
[0184] The reaction of the lip mucosa was then scored by 2
independent observers according to the scoring system developed by
Parkins et al. (Parkins et al, Radiotherapy & Oncology, 1:
165-173, 1983), in which the erythema status as well as the
presence of edema, desquamation and exudation was quoted.
Additionally, animals were weighed before each recording of their
erythema/edema status.
[0185] FIG. 12A: illustrated the weight of the mice following
irradiation. Values are reported to the initial weight of the mice
that was set to 100. CTRL: control mice injected with 30 .mu.l of a
saline solution. n=2 for each values reported, S.D. are indicated.
x values are days
[0186] FIG. 12B is illustrative of the erythema/edema scoring
following irradiation. The edema and erythema status of the ventral
lip of the same mice as in FIG. 12A was quantified. n=2 for each
value reported. x values are days The results of these experiments
indicate that the TAT-IB Peptides can protect against weight loss
and erythema/edema associated with ionizing radiation.
Example 13
Suppression of JNK Transcription Factors by L-TAT-IB1 Peptides
[0187] Gel retardation assays were carried out with an AP-1 doubled
labeled probe (5'-CGC TTG ATG AGT CAG CCG GAA-3'. HeLa cell nuclear
extracts that were treated or not for one hour with 5 ng/ml
TNF-.alpha., as indicated. TAT and L-TAT-IB1 peptides were added 30
minutes before TNF-.alpha.. Only the part of the gel with the
specific AP-1 DNA complex (as demonstrated by competition
experiments with non-labeled specific and non-specific competitors)
is shown. L-TAT-IB1 peptides decrease the formation of the AP-1 DNA
binding complex in the presence of TNF-.alpha.. (See, FIG. 11).
Example 14
Protection Against Noise-Induced Hearing Loss by D-TAT-IB
Peptides
[0188] A solution of D-JNKI (1 uM, 1 ul/hr) was injected into the
right internal ear of a guinea pig as shown in FIG. 13, panel A,
whereas the left ear was injected with saline only. The pig was
then exposed to a noise trauma (120 db, 30 minutes), and recording
of hearing sensitivity was performed three days after (FIG. 13,
panel B) as well as histological examination of the inner ear (FIG.
13, panel C an D). As shown in FIG. 13 the ciliated structures on
the JNKI treated ear are completely protected from noise induced
destruction as judged from the histological examination, in
contrast to the non-treated ear where most of the ciliated
structures have disappear. Furthermore, the sensitivity of the
D-JNK1 treated ear to noise appear to be preserved (FIG. 13, panel
B).
Example 15
Protection Against Antibiotic-Induced Hearing Loss by D-TAT-IB
Peptides
[0189] Chicken internal ears were treated with streptomycin in the
presence/absence of D_JNKI. TUNEL experiments were then performed
to detect apoptosis (green nuclei). As shown in FIG. 14, D-JNKI
fully protects internal ears from streptomycin induced apoptosis.
Thus D-JNK-I is useful in the prevention of hearing loss conditions
sustained by antibiotic therapy.
Example 16
Protection Against Pancreatic Islet Destruction Induced by
Proinflammatory Cytokines by D-TAT-IB Peptides
[0190] Pancreatic islets cells were treated with D-JNK1 (1 mM for
one hour before being exposed to interleukin 1B (10 ng/ml). As
shown in FIG. 15, D-JNK1 treated islets resist IL-1B induced
destruction. This indicates that treatment with D-JNK1 helps
preserve grafted islets.
Example 17
Increase Recovery of Pancreatic Islets Cells by D-TAT-IB
Peptides
[0191] D-JNK-I were added together with collagenase during islet
cell isolation. This resulted in an increased yield of islet after
3 days in culture as measured by the increase in lactate
dehydrogenase. See FIG. 15.
Example 18
General Methods Used in Testing the Effects of JNKI Peptides on JNK
Activation and JNK-Related Action
[0192] General Neuronal Culture: Small pieces of cortex from the
brains of two day old rat pups were dissected and incubated with
200 units of papain for 30 minutes at 34.degree. C. Then, the
neurons were plated at densities of approximately 1.times.10.sup.6
cells/plate on dishes that had been pre-coated with 100 .mu.g/mL
poly-D-lysine. The cells were cultured using a B27/Neurobasal (Life
Technologies) culture medium, supplemented with 0.5 m glutamine,
100 U/mL penicillin and 100 .mu.g/mL streptomycin.
[0193] Lactate dehydrogenase (LDH) cytotoxicity assay: The amount
of LDH released into the culture medium was measured using the
Cytotox 96 non-radioactive cytotoxicity assay kit (Promega).
[0194] GST-c-Jun pull-down and kinase assay: Cellular extracts were
prepared by scraping cells in lysis buffer (20 mM Tris-acetate, 1
mM EGTA, 1% Triton X-100, 10 mM p-nitrophenyl-phosphate, 5 mM
sodium pyrophosphate, 10 mM .beta.-glycerophosphate, 1 mM
dithiothreitol). 25 .mu.g samples were incubated for 1 hour at room
temperature with 1 .mu.g GST-c-Jun (amino acid residues 1-89) and
10 .mu.L glutathione-agarose beads (Sigma). The beads were washed
four times and then resuspended in the lysis buffer described
above. In vitro kinase assays were then performed using recombinant
JNK1.alpha.1 and 0.5 .mu.g of a substrate selected from the group
consisting of GST-fusion proteins (e.g, GST-Jun and GST-Elk1 fusion
proteins), casein and histone (Sigma). Reactions were initiated
with 10 mM MgCl.sub.2 and 10 .mu.M ATP in the presence of 5 .mu.Ci
.sup.33P-ATP, and were incubated for 30 minutes at 30 degrees
Celsius. The reaction products were separated by SDS-PAGE, and the
gels were dried and then exposed to X-ray films (Kodak).
[0195] Western blots: Total protein extracts were obtained by
scraping cells in lysis buffer (described above), separating the
proteins on a 12% SDS polyacrylamide gel. The separated proteins
were then transferred onto polyvinylidene fluoride (PVDF) membrane.
Antibodies used in the Western blots described herein were obtained
from Alexis.
[0196] Separation of nuclei from cytoplasm: To isolate nuclei for
Western blot analysis (see FIG. 17B), neurons were lysed for 15
minutes in lysis buffer, and then the samples were centrifuged at
300 g for 10 minutes at 4.degree. C. The nuclear pellets were
reconstituted in lysis buffer and then sonicated.
[0197] Real-time RT-PCR: Real-time RT-PCR was performed using
specific primers on a lightcycler apparatus (Roche). The
housekeeping actin transcript was used to normalize for the amount
and quality of the RNAs that were extracted by the Chomczynski
method. See Chomczynski et al., Anal. Biochem., 162:156-59 (1987),
hereby incorporated by reference in its entirety. The sequences of
the primers used were as follows:
2 c-Fos: Forward: 5'-GCTGACAGATACACTCCAAG-3' Reverse:
5'-CCTAGATGATGCCGGAAACA-3' Actin: Forward:
5'-AACGGCTCCGGCATGTGCAA-3' Reverse: 5'-ATTGTAGAAGGTGTGGTGCCA-5'
[0198] P-c-jun immunohistochemistry: P-c-jun, as used herein refers
to phosphorylated forms of c-jun. P-c-jun was targeted with a
rabbit polyclonal antibody (500.times. in PBS) (Cell Signaling
Technology). The resulting antibody complex was visualized with
3,3-diaminobenzidine as the substrate.
[0199] Transient ischemia in adult mice: Using male ICR-CD1 mice
(approximately 6 weeks old and weighing in the range of about 18 to
37 g) (Harlan, Inc.), ischemia was provoked by introducing a
filament from the common carotid artery into the internal carotid
artery and advancing the filament into the arterial circle, thereby
occluding the middle cerebral artery. See e.g., Huang et al.,
Science, 265:1883-85 (1994); Hara et al., Proc. Natl. Acad. Sci.
(USA) 94:2007-12 (1997); each of which is hereby incorporated by
reference in its entirety. Regional cerebral blood flow was
measured by laser-Doppler flowmetry with a probe fixed on the skull
throughout the ischemia and until 10 minutes after reperfusion.
Rectal temperature was measured and maintained at 37.degree. C. The
animals were sacrificed 48 hours after reperfusion. Serial cryostat
sections 20 .mu.M-thick were traced using a computer-microscope
system equipped with the Neurolucida program (Microbrightfield,
Inc.), and the volumes of the ischemic area and of the whole brain
were calculated (blind) with the Neuroexplorer program. Systolic
and diastolic blood pressure were measured with an arterial
catheter in three additional mice from 10 minutes before the
D-JNKI1 injection until 30 minutes afterwards. These blood pressure
measurements showed that the injections did not affect blood
pressure (i.e., less than 10% change). The Guidelines of the Swiss
Federal Veterinary Office were followed in all experiments.
[0200] Permanent focal ischemia in young (P14) rats: Middle
cerebral artery occlusion was obtained by electrocoagulating the
middle cerebral artery at a position closed to its origin at the
junction with the olfactory branch. The rats (from Wistar), which
weighed in the range of about 27-35 g, were sacrificed 24 hours
after middle cerebral artery occlusion. The rats were sacrificed
using an overdose of chloral hydrate and were perfused through the
left ventricle with Zamboni's fixative. The brains were postfixed
for 2 hours in the same solution used for perfusion, and then the
brains were infiltrated overnight in 30% sucrose for
cryoprotection. The outlines of each ischemic area were drawn on
(stained) with a computer-microscope system. The area of the
ischemic lesion and of the whole brain were traced from 50 .mu.m
serial cryostat sections stained with cresyl violet using the
Neurolucida program, and the volumes of each were calculated using
the Neuroexplorer program, as described above.
[0201] Statistics: Data from both ischemia models (i.e., transient
and permanent) were transformed logarithmically to satisfy the
Gaussian criterion. Data was analyzed with an overall ANOVA
(p<0.0001 for both models) followed by one-tailed unpaired
t-tests.
Example 19
Sensitivity and Specificity of JNKI Peptides Against JNK Action
[0202] The JNKI1 peptides used in these experiments are aimed at
blocking the access of JNK to c-Jun and other substrates by a
direct competitive mechanism. See e.g., Bonny et al., Diabetes,
50:77-82 (2001); Barr et al., J. Biol. Chem., 277:10987-97 (2002),
each of which is hereby incorporated by reference in its
entirety.
[0203] The inhibitory effect of L-JNKI1 and D-JNKI1 on JNK
activation and action was tested using the kinase assays, as
described above in Example 18. The results of these experiments are
shown in FIGS. 16A-16C. The inhibitory effect of L-JNKI1 and
D-JNKI1 on JNK activation and action is shown by their ability to
prevent the phosphorylation in vitro of known JNK targets c-Jun and
Elk1 using JNK1.alpha.1. (See FIG. 16A). The terms "P-Jun" and
"P-Elkl," as used herein, refer to the radiolabeled (i.e.,
phosphorylated with .sup.33P-ATP) forms of GST-Jun and GST-Elk1
substrates, respectively. FIG. 16B demonstrates the inhibitory
effect of the 20 amino acid minimal JNK-inhibitory sequence of
JIP-IB1 (L-form of JBD.sub.20 (SEQ ID NO:21)) in dose response
experiments, using conditions similar to those used to test the
inhibitory effect of L-JNKI1 and D-JNKI1 and using decreasing
amounts of L-JBD.sub.20. FIG. 16B illustrates that the L-JBD.sub.20
peptide (SEQ ID NO:21) alone (i.e., without the TAT sequence) can
inhibit JNK action. JBD.sub.20 was also shown to inhibit other JNK
targets including ATF2, IRS-1, MADD, bcl-xl. In each of these
cases, the IC.sub.50 was about 1 .mu.M (data not shown). The TAT
sequence was not linked to JBD.sub.20 in these experiments,
because, at concentration greater than 50 .mu.M, the TAT sequence
induces a nonspecific precipitation of the proteins in the
extracts. Below 50 .mu.M, TAT does not influence the inhibitory
properties of the JBD.sub.20 peptides.
[0204] In vitro experiments were performed to determine the
specificity of the JNKI peptides in blocking JNK activation. In
particular, the effect of these peptides on the activity of 40
different kinases (10 .mu.M peptides, 10 .mu.M ATP) towards their
respective substrates was tested. The complete list of substrates
used in these experiments can be found at
http://www.upstate.com/img/pdf/KinaseProfiler.pdf. As expected, the
JNKI peptides had an affect on the JNKs and MKK4 and MKK7 kinases,
all of which contain JNK-binding domains. The peptides (both the
L-JNKI1 and D-JNKI1 forms) completely failed to interfere with the
activities of all other kinases. Additional experiments showed that
500 .mu.M of the JBD.sub.20 peptides did not interfere with the
activity of 6 particular kinases: ERK2, p38, pKC, p34, caK and pKA
(FIG. 16C). The substrates for these kinases are ERK2:ERK1;
p38:ATF2; p34, pKC, pKA:histone; and caK:caseine. This level of
specificity is far above those achieved with other small chemical
inhibitors of Jun-N-terminal kinase, thereby demonstrating the
extremely high selectivity of the JNKI peptides of the invention.
For a discussion of other small chemical inhibitors of the Jun
N-terminal kinase (JNK), see Bennett et al., Proc. Natl. Acad. Sci.
(USA), 98:13681-86 (2001), hereby incorporated by reference in its
entirety.
Example 20
Effects of the JNKI Peptides on JNK Targets Inside NMDA-Treated
Cortical Neurons
[0205] A series of experiments were performed to analyze the
effects of the JNKI peptides of the invention on different JNK
targets inside neurons. The activation of JNK in
N-methyl-D-aspartate (NMDA)-treated cortical neurons in culture was
estimated by performing kinase assays on pulled-down JNK using
GST-c-Jun, using the methods described above. (See e.g., Ko et al.,
J. Neurochem. 71:1390-1395 (1998); Coffey et al., J. Neurosci.
20:7602-7613 (2000), each of which is hereby incorporated in its
entirety by reference). The results of these experiments are shown
in FIGS. 17-18.
[0206] FIG. 17A shows the JNK activity in untreated neurons ("0"),
after 10 minutes exposure to 100 .mu.M NMDA (10') or after 30
minutes exposure to 100 .mu.M NMDA. The two lanes at the right of
FIG. 17C demonstrate that JNK activation was essentially unchanged
by D-INKI1. The increase in JNK activity appeared maximal (i.e.,
2.2 fold) after 30 minutes of NMDA treatment (FIG. 17A). This
increase in JNK activity translated into an elevated c-Jun
phosphorylation (FIG. 17B). Addition of the cell-penetrating
peptides L-JNKI1 and D-JNKI1 was shown to completely prevent the
increase in P-c-Jun after 5 hours of exposure to 100 .mu.M NMDA,
despite a normal level of JNK activation. Addition of L-JNKI1 and
D-JNKI1 brought the level of P-c-Jun below even the level of
P-c-Jun in the control.
[0207] NMDA-induced transcription of the c-fos gene, under the
influence of JNK via the Elk1 transcription factor was also
completely prevented by the addition of L-JNKI and D-JNKI1 (FIG.
17C). c-fos expression was quantitated by real-time PCR
(Lightcycler) using RNA extracted using the methods described above
in Example 18. The data in FIG. 17C is presented as c-fos
expression relative to actin (n=4). For a description of the
induction of c-fos expression through JNK-mediated TCF/Elk-1
phosphorylation, see Cavigelli et al., EMBO J., 14:5957-5964
(1995), hereby incorporated by reference in its entirety.
[0208] The time course of NMDA neurotoxicity and neuroprotection by
L-JNKI1 and D-JNKI1, as well as two control peptides, TAT-empty
(i.e., the TAT sequence alone, without the JBD.sub.20 sequence) and
L-JNKI1.sub.mut (having six amino acids mutated to alanine, as
described in Bonny et al., Diabetes 50:77-82 (2001), hereby
incorporated by reference in its entirety). The micrographs of FIG.
18 show Hoechst-stained neurons at 24 hours after treatment.
Addition of the L-JNKI and D-JNKI1 peptides completely protected
neurons against the excitotoxic effects of NMDA (FIG. 18) or
kainate (data not shown), while the addition of control peptides
had no neuroprotective effect. At 12 hours post-treatment, both
L-JNKI1 and D-JNKI1 peptides were shown to inhibit neuronal death
whereas TAT-empty peptides had no effect (FIG. 18).
[0209] As seen in FIG. 18, the D-form of the cell-penetrating
peptides of the invention, i.e., D-JNKI1, was superior in
protecting neurons for extended periods of time, i.e., 12 hours, 24
hours and 48 hours post-exposure to 100 .mu.M NMDA. These
micrographs indicate that at 24 hours post-treatment, D-JNKI1 still
gave total neuroprotection, as the control cultures and the
cultures treating with D-JNKI1 and NMDA were comparable. The L-form
of JNKI1 no longer protected the neurons at 24 hours
post-treatment, presumably because the L-forms of peptides are
generally more susceptible to degradation. The TAT-empty peptides
did not affect cell death in any conditions. The histogram in FIG.
18 depicts the level of neuronal death at 12, 24 and 48 hours after
exposure to 100 .mu.M NMDA, as indicated by LDH activity in the
medium of the Petri dish. Absorbance values, which represent the
LDH concentration, have been converted into % neuronal death values
by dividing the absorbance values by the average absorbance for
total LDH. The average absorbance for total LDH was obtained from
the medium plus lysed neurons.
Example 21
In Vivo Delivery of Cell-Permeable JNKI Peptides
[0210] To test the feasibility of using the cell-permeable peptides
in in vivo applications, their ability to penetrate into the brain
was evaluated using FITC-labeled L-JNKI1 and D-JNKI1. For a
discussion on the in vivo delivery of a biologically active protein
into a mouse, see Schwarze et al., Science, 285:1569-72 (1999),
hereby incorporated by reference in its entirety. These experiments
showed that both FITC-labeled L-JNKI1 and D-JNKI1 were able to
cross the blood-brain barrier and penetrate into the neurons of
adult mice and rats of various ages. Both FITC-labeled L-JNKI1 and
D-JNKI1 were able to penetrate into the neurons within 1 hour of
intraperitoneal injection (data not shown).
Example 22
Neuroprotection by the JNKI Peptides Against Transient and
Permanent Focal Cerebral Ischemia
[0211] In a model of mild ischemia in mice, the left middle
cerebral artery was occluded for 30 minutes, followed by 48 h of
reperfusion. The control vehicle-treated group received an
injection of phosphate buffer saline (PBS) only. In the control
vehicle-treated group, this occlusion resulted systematically in a
major infarction containing severely pyknotic cells, which were
predominantly found in the cortex and the stratum in all brains,
and in 7 of the brains, these cells were also found in the
hippocampus. The mean infarction volume was 67.4 mm.sup.3 (n=12) in
those subjects in the control vehicle-treated group.
[0212] To evaluation the efficacy and "therapeutic window" of
treatment (i.e., the timeframe following injury during which
treatment with the peptides of the invention remains effective),
subjects were treated with intracerebro-ventricular (icv) injection
of D-JNKI1 (15.7 ng in 2 .mu.L of PBS). FIG. 19A demonstrates
cresyl violet-stained sections that show typical examples of the
resulting infarct (bar, 1 mm). FIG. 19B depicts infarction volumes
following icv injection of D-JNKI1 at different times before (-1
hour) or after (+3, 6, or 12 hours) after middle cerebral artery
occlusion. In FIG. 19B, an asterisk (*) indicates the result is
statistically different from the control (as indicated by a
t-test).
[0213] Pretreatment 1 hour before middle cerebral artery occlusion
with the icv injection of D-JNKI1 significantly decreased the
infarct volume measured 48 hours after reperfusion by 88%, to a
volume of 7.8 mm. (FIGS. 19A-19B). Administering the D-JNKI1
peptide 3 or 6 hours after middle cerebral artery occlusion was
still potently protective, as the mean infarct volume for subjects
injected 3 hours post-occlusion was reduced to 5.8 mm.sup.3 (a
reduction of 91% compared to untreated animals), and the mean
infarct volume for subjects injected 6 hours post-occlusion was
reduced to 4.8 mm (a reduction of 93% compared to untreated
animals). In contrast, D-JNKI1 peptide injection at 12 hours after
middle cerebral artery occlusion was not significantly protective.
To confirm the achievement of complete ischemia followed by
reperfusion was confirmed in all animals by monitoring regional
cerebral blood flow in the territory of the left middle cerebral
artery.
[0214] The protective abilities of D-JNKI1 against permanent focal
ischemia in young (P14) rats was also evaluated. An ischemic zone
in the cerebral cortex of P14 rats by performing a permanent
occlusion of the middle cerebral artery, thereby inducing a zone of
massive degeneration restricted to the parietotemporal cortex. As
brain volumes in the P14 rats were variable, the lesions were
expressed as a percentage of the volume of the cerebral hemisphere.
D-JNKI1 was injected intraperitoneally at a concentration of 11
mg/kg, which corresponds to approximately 340 .mu.g. D-JNKI1 was
administered 30 minutes prior to middle cerebral artery occlusion,
or 6 or 12 hours post-occlusion. The rats were fixed at 24 hours
post-occlusion. At each of these time-points (i.e., administration
at -30 minutes, +6 h or +12 h), D-JNKI1 caused major and
statistically significant decreases in the infarct volume, as
compared to control animals (FIGS. 20A-20B). Administration of
D-JNKI1 30 minutes prior to occlusion led to a decrease in the
infarct volume of 68%, while peptide administration at 6 and 12
hours post-occlusion led to decreases in infarct volume of 78% and
49%, respectively.
[0215] Immunohistochemistry analysis was performed to determine the
activation of the c-Jun transcription factor, a major target of
JNK, in the brains of rat pups with permanent ischemia.
Phosphorylation of c-Jun was evident in many neurons in the
peri-infarcted cortex (FIG. 5C, bar=200 .mu.M). In contrast, in
brains treated with D-JNKI1 peptide, the peri-infarcted cortex was
negative, and only a few positive neurons at the border of the
infarcted region were detected.
Example 22
Behavioral Evaluation of Potential Side-Effects of JNKI
Peptides
[0216] Typically, the high toxicity of other neuroprotective
compounds has severely limited their clinical use. (See Gladstone
el al., Stroke, 33:2123-36 (2002), incorporated by reference in its
entirety). The ability of mice to maintain themselves on horizontal
turning rotarod was used as criterion for possible side effects of
different doses of D-JNKI 1 and of a therapeutic dose of MK-801 (1
mg/Kg, a standard therapeutic dose). In particular, the motor
function of the mice was evaluated using the rotarod test at 3 h,
24 h, 6 days and 12 days after both i.p. (11 and 110 mg/Kg) and icv
injections of D-JNKI1 (2 .mu.l containing 15.7 ng or 157 ng of
D-JNKI1). The i.p. injection of MK-801 (1 mg/Kg) was used as a
control compound during this assessment procedure.
[0217] The mice were trained the day before and in the morning of
the experimental day, in order to reduce the variability between
subjects. Both training and test sessions were identical for
control and injected mice. The motor function of each mouse was
examined immediately before the injection and at 1, 6 and 12 days
after the injection. The mice were placed on the rotarod, which was
programmed to accelerate uniformly from 4 to 40 rpm. The latency to
falls for each mouse tested was recorded. The results of this
assessment using the rotarod method are presented in Table 2 as
median latency to fall (measured in seconds).
3TABLE 2 EFFECT OF D-JNKI1 ON MOTOR COORDINATION Median latency to
fall (secs) DOSE -1 h +3 h 1 day 6 days 12 days PBS 2 .mu.l icv 234
202 238 268 246 MK-801 1 mg/Kg 226 incapable 174 233 292 i.p.
D-JNKI1 11 mg/Kg 204 221 372 287 418 i.p. 110 mg/Kg 276 266 447 416
325 i.p. 15.7 ng icv 210 342 302 345 285 157 ng icv 260 200 253 338
335 2 .mu.l PBS 234 202 238 268 246 icv
[0218] As seen in Table 2, motor coordination was found to be
unimpaired with both the i.p. and icv D-JNKI1doses (i.e., both the
dose, 2.8 .mu.l/Kg, that conferred 90% neuroprotection, and a
10-fold higher dose). In contrast, MK-801 led to a dramatic
impairment of motor coordination, as the mice were unable to stand
on the rotor wheel. (See e.g., Table 2; Dawson et al., Brain Res.
892:344:350 (2001) (describing similar results for other
neuroprotectants), and a 10-fold higher dose of MK-801 killed all
the mice. The side effects of the lower dose of MK-801 were found
to essentially disappear after 24 hours. At 6 and 15 days following
treatment with D-JNKI1, no sign of motor impairment was found, and
the rotarod scores were reproducibly better than in the control
mice.
[0219] Equivalents
[0220] From the foregoing detailed description of the specific
embodiments of the invention, it should be apparent that unique
cell-permeable bioactive peptides have been described. Although
particular embodiments have been disclosed herein in detail, this
has been done by way of example for purposes of illustration only,
and is not intended to be limiting with respect to the scope of the
appended claims which follow. In particular, it is contemplated by
the inventor that various substitutions, alterations, and
modifications may be made to the invention without departing from
the spirit and scope of the invention as defined by the claims.
Sequence CWU 1
1
33 1 23 PRT Artificial Sequence chemically synthesized 1 Asp Thr
Tyr Arg Pro Lys Arg Pro Thr Thr Leu Asn Leu Phe Pro Gln 1 5 10 15
Val Pro Arg Ser Gln Asp Thr 20 2 21 PRT Artificial Sequence
chemically synthesized 2 Glu Glu Pro His Lys His Arg Pro Thr Thr
Leu Arg Leu Thr Thr Leu 1 5 10 15 Gly Ala Gln Asp Ser 20 3 23 PRT
Artificial Sequence chemically synthesized 3 Thr Asp Gln Ser Arg
Pro Val Gln Pro Phe Leu Asn Leu Thr Thr Pro 1 5 10 15 Arg Lys Pro
Arg Tyr Thr Asp 20 4 21 PRT Artificial Sequence chemically
synthesized 4 Ser Asp Gln Ala Gly Leu Thr Thr Leu Arg Leu Thr Thr
Pro Arg His 1 5 10 15 Lys His Pro Glu Glu 20 5 77 PRT Artificial
Sequence VARIANT (36) Wherein Xaa is any amino acid as defined in
the specification. 5 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Arg Pro 20 25 30 Thr Thr Leu Xaa Leu Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Gln Asp Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 6 19 PRT
Artificial Sequence chemically synthesized 6 Xaa Xaa Asp Gln Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Leu Thr Thr 1 5 10 15 Pro Arg Xaa 7
10 PRT Artificial Sequence chemically synthesized 7 Gly Arg Lys Lys
Arg Arg Gln Arg Arg Arg 1 5 10 8 10 PRT Artificial Sequence
chemically synthesized 8 Arg Arg Arg Gln Arg Arg Lys Lys Arg Gly 1
5 10 9 17 PRT Artificial Sequence Description of Artificial
Sequence Chemically Synthesized 9 Xaa Xaa Xaa Xaa Arg Lys Lys Arg
Arg Gln Arg Arg Arg Xaa Xaa Xaa 1 5 10 15 Xaa 10 17 PRT Artificial
Sequence chemically synthesized 10 Xaa Xaa Xaa Xaa Arg Arg Arg Gln
Arg Arg Lys Lys Arg Xaa Xaa Xaa 1 5 10 15 Xaa 11 35 PRT Artificial
Sequence chemically synthesized 11 Gly Arg Lys Lys Arg Arg Gln Arg
Arg Arg Pro Pro Asp Thr Tyr Arg 1 5 10 15 Pro Lys Arg Pro Thr Thr
Leu Asn Leu Phe Pro Gln Val Pro Arg Ser 20 25 30 Gln Asp Thr 35 12
33 PRT Artificial Sequence chemically synthesized 12 Gly Arg Lys
Lys Arg Arg Gln Arg Arg Arg Pro Pro Glu Glu Pro His 1 5 10 15 Lys
His Arg Pro Thr Thr Leu Arg Leu Thr Thr Leu Gly Ala Gln Asp 20 25
30 Ser 13 42 PRT Artificial Sequence chemically synthesized 13 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10
15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Pro Thr Thr Leu Xaa Leu Xaa
20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Gln Asp Xaa Xaa 35 40 14 35 PRT
Artificial Sequence chemically synthesized 14 Thr Asp Gln Ser Arg
Pro Val Gln Pro Phe Leu Asn Leu Thr Thr Pro 1 5 10 15 Arg Lys Pro
Arg Tyr Thr Asp Pro Pro Arg Arg Arg Gln Arg Arg Lys 20 25 30 Lys
Arg Gly 35 15 33 PRT Artificial Sequence chemically synthesized 15
Ser Asp Gln Ala Gly Leu Thr Thr Leu Arg Leu Thr Thr Pro Arg His 1 5
10 15 Lys His Pro Glu Glu Pro Pro Arg Arg Arg Gln Arg Arg Lys Lys
Arg 20 25 30 Gly 16 42 PRT Artificial Sequence checmically
synthesized 16 Xaa Xaa Asp Gln Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa
Leu Thr Thr 1 5 10 15 Pro Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg
Arg Arg Gln Arg Arg 20 25 30 Lys Lys Arg Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 35 40 17 29 PRT Artificial Sequence chemically synthesized 17
Pro Gly Thr Gly Cys Gly Asp Thr Tyr Arg Pro Lys Arg Pro Thr Thr 1 5
10 15 Leu Asn Leu Phe Pro Gln Val Pro Arg Ser Gln Asp Thr 20 25 18
27 PRT Artificial Sequence chemically synthesized 18 Ile Pro Ser
Pro Ser Val Glu Glu Pro His Lys His Arg Pro Thr Thr 1 5 10 15 Leu
Arg Leu Thr Thr Leu Gly Ala Gln Asp Ser 20 25 19 29 PRT Artificial
Sequence chemically synthesized 19 Gly Ala Tyr Gly Tyr Ser Asn Pro
Lys Ile Leu Lys Gln Ser Met Thr 1 5 10 15 Leu Asn Leu Ala Asp Pro
Val Gly Asn Leu Lys Pro His 20 25 20 29 PRT Artificial Sequence
chemically synthesized 20 Thr Asn Glu Asp His Leu Ala Val His Lys
His Lys His Glu Met Thr 1 5 10 15 Leu Lys Phe Gly Pro Ala Arg Asn
Asp Ser Val Ile Val 20 25 21 20 PRT Artificial Sequence Description
of Artificial Sequence chemically synthesized 21 Arg Pro Lys Arg
Pro Thr Thr Leu Asn Leu Phe Pro Gln Val Pro Arg 1 5 10 15 Ser Gln
Asp Thr 20 22 20 PRT Artificial Sequence Description of Artificial
Sequence chemically synthesized 22 Thr Asp Gln Ser Arg Pro Val Gln
Pro Phe Leu Asn Leu Thr Thr Pro 1 5 10 15 Arg Lys Pro Arg 20 23 32
PRT Artificial Sequence Description of Artificial Sequence
chemically synthesized 23 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg
Pro Pro Arg Pro Lys Arg 1 5 10 15 Pro Thr Thr Leu Asn Leu Phe Pro
Gln Val Pro Arg Ser Gln Asp Thr 20 25 30 24 32 PRT Artificial
Sequence Description of Artificial Sequence chemically synthesized
24 Thr Asp Gln Ser Arg Pro Val Gln Pro Phe Leu Asn Leu Thr Thr Pro
1 5 10 15 Arg Lys Pro Arg Pro Pro Arg Arg Arg Gln Arg Arg Lys Lys
Arg Gly 20 25 30 25 34 PRT Artificial Sequence VARIANT (1)..(4)
Wherein Xaa is any amino acid as defined in the specification 25
Xaa Xaa Xaa Xaa Arg Lys Lys Arg Arg Gln Arg Arg Arg Xaa Xaa Xaa 1 5
10 15 Xaa Arg Pro Thr Thr Leu Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Gln 20 25 30 Asp Xaa 26 34 PRT Artificial Sequence VARIANT (1)
Wherein Xaa is Ser or Thr 26 Xaa Asp Gln Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Leu Xaa Leu Thr Thr Pro 1 5 10 15 Arg Xaa Xaa Xaa Xaa Arg Arg
Arg Gln Arg Arg Lys Lys Arg Xaa Xaa 20 25 30 Xaa Xaa 27 20 PRT
Artificial Sequence Description of Artificial Sequence chemically
synthesized 27 Arg Pro Lys Arg Pro Thr Ala Ala Asn Ala Phe Pro Gln
Val Pro Arg 1 5 10 15 Ser Gln Asp Thr 20 28 20 PRT Artificial
Sequence Description of Artificial Sequence chemically synthesized
28 Thr Asp Gln Ser Arg Pro Val Ala Pro Phe Ala Asn Ala Ala Thr Pro
1 5 10 15 Arg Lys Pro Arg 20 29 21 DNA Artificial Sequence
Description of Artificial Sequence oligonucleotide probe 29
cgcttgatga gtcagccgga a 21 30 20 DNA Artificial Sequence
Description of Artificial Sequence oligonucleotide primer 30
gctgacagat acactccaag 20 31 20 DNA Artificial Sequence Description
of Artificial Sequence oligonucleotide primer 31 cctagatgat
gccggaaaca 20 32 20 DNA Artificial Sequence Description of
Artificial Sequence oligonucleotide primer 32 aacggctccg gcatgtgcaa
20 33 21 DNA Artificial Sequence Description of Artificial Sequence
oligonucleotide primer 33 attgtagaag gtgtggtgcc a 21
* * * * *
References