Cell-Permeable Peptide Inhibitors of the JNK Signal Transduction Pathway

Abstract

Protein kinase inhibitors and more specifically inhibitors of the protein kinase c-Jun amino terminal kinase are herein described. Additionally, JNK inhibitor sequences, chimeric peptides, nucleic acids encoding same as well as pharmaceutical compositions for treating pathophysiologies associated with JNK signaling are herein provided.

Claims

1. JNK inhibitor sequence comprising less than 150 amino adds in length, wherein the inhibitor sequence comprises or consists of at least one amino acid sequence according to SEQ ID NOs: 1, 2, 3 or 4, or a fragment, derivative or variant thereof.

2. The JNK inhibitor sequence according to claim 1, wherein the JNK inhibitor sequence comprises a range of 5 to 150 amino acid residues, more preferably 10 to 100 amino acid residues, even more preferably 10 to 75 amino acid residues and most preferably a range of 15 to 50 amino acid residues.

3. The JNK inhibitor sequence of claim 1, wherein the JNK inhibitor sequence binds c-jun amino terminal kinase (JNK).

4. The JNK inhibitor sequence of claim 1, wherein the JNK inhibitor sequence inhibits the activation of at least one JNK targeted transcription factor when the JNK inhibitor sequence is present in a JNK expressing cell.

5. The JNK inhibitor sequence of claim 1, wherein the JNK targeted transcription factor is selected from the group consisting of c-Jun, ATF2, and Elk I.

6. The JNK inhibitor sequence of claim 1, wherein the JNK inhibitor sequence alters a JNK effect when the peptide is present in a JNK expressing cell.

7. A chimeric peptide comprising at least one first domain and at least one second domain linked by a covalent bond, the first domain comprising a trafficking sequence, and the second domain comprising a JNK inhibitor sequence.

8. The peptide of claim 7, wherein the trafficking sequence comprises the amino acid sequence of a human immunodeficiency virus TAT polypeptide.

9. The peptide of claim 7, wherein the trafficking sequence comprises the amino acid sequence of SEQ ID NO: 5, 6, 7 or 8.

10. The peptide of claim 7, wherein the trafficking sequences augments cellular uptake of the peptide.

11. The peptide of claim 7, wherein the trafficking sequence directs nuclear localization of the peptide.

12. The peptide of claim 7, wherein the inhibitor sequence comprises or consists of at least one amino acid sequence according to SEQ ID NOs: 1, 2, 3 or 4, or a fragment, derivative or variant thereof.

13. The peptide of claim 7, wherein the peptide comprises the amino acid sequence of any of SEQ ID NOs: 9, 10, 11 or 12, or a fragment, or variant thereof.

14. An isolated nucleic acid encoding a JNK inhibitor sequence of claim 1.

15. A vector comprising the nucleic acid of claim 14.

16. A cell comprising the vector of claim 15.

17. An antibody which binds immunospecifically to a JNK inhibitor sequence according of claim 1.

18. A pharmaceutical composition comprising a JNK inhibitor sequence of claim 1, and a pharmaceutically acceptable carrier.

19. Use of a JNK inhibitor sequence of claim 1, for the preparation of a pharmaceutical composition for treating a pathophysiology associated with activation of JNK in a subject.

20. Use according to claim 19, wherein the pathophysiology is selected from malignancies of lung, breast, lymphoid, gastrointestinal, and genito-urinary tract as well as adenocarcinomas, including malignancies such as colon cancers, renal-cell carcinoma, prostate cancer, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus, as well as leukemia, disorders or pathophysiologies associated with oncogenic transformation as well as cancers with Bcr-Abl oncogenic transformations, non-malignant or immunological-related cell proliferative diseases selected from psoriasis, pemphigus vulgaris, Behcet's syndrome, acute respiratory distress syndrome (ARDS), ischemic heart disease, post-dialysis syndrome, rheumatoid arthritis, acquired immune deficiency syndrome, vasculitis, septic shock, pathophysiologies associated with activation of JNK in a cell selected from restenosis, hearing loss, ear trauma, ischemia, stroke and/or disorders or pathophysiologies associated with maturation and differentiation of immune cells, reperfusion injuries, hypoxia, apoptosis-related, response to stressful stimuli, and with secondary effects due to treatment with proinflammatory cytokines, effects associated with diabetes or with cellular shear stress, selected from pathological states induced by arterial hypertension, including heart and cardiac hypertrophy and arteriosclerotic lesions, and at bifurcations of blood vessels, by ionizing radiation as used in radiotherapy and ultraviolet light (UV lights), by free radicals, DNA damaging agents, including chemotherapeutic drugs, by ischemia/reperfusion injuries, by hypoxia, by hypo- and hyperthermia, or to inhibit inflammatory, autoinflammatory, immune and autoimmune diseases, degenerative diseases, myopathies, cardiomyopathies, and graft rejection.

21.-23. (canceled)

Description

DESCRIPTION OF FIGURES

[0131] FIGS. 1A-IC are diagrams showing alignments of conserved JBD domain regions in the indicated transcription factors. JNK inhibitor sequences were identified by inspecting these sequence alignments. The results of this alignment are exemplarily shown in FIGS. 1A-1C. FIG. 1A depicts the region of highest homology between the JBDs of IB1 (SEQ ID NO: 13), IB2 (SEQ ID NO: 14), c-Jun (SEQ ID NO: 15) and ATF2 (SEQ ID NO: 16). FIG. 1B depicts the amino acid sequence of the JBDs of L-IB1(s) (SEQ ID NO: 1) and L-IB1 (SEQ ID NO: 17) for comparative reasons. 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 (SEQ ID NOS 5, 9, 23, 6, 11 and 25, respectively, in order of appearance) that include a JNK inhibitor sequence and a trafficking sequence. In the example shown, the trafficking sequence is derived from the human immunodeficiency virus (HIV) TAT polypeptide, and the JNK inhibitor sequence is derived from an IB1(s) polypeptide. Human, mouse, and rat sequences are identical in FIG. 1B and FIG. 1C.

[0132] FIG. 2 is a diagram showing sequences of generic TAT-IB fusion peptides from human, mouse and rat (SEQ ID NOS 21, 149, 24, 22, 150 and 26, respectively, in order of appearance).

[0133] FIG. 3 depicts the results from the evaluation of the neuroprotection against focal cerebral ischemia in a permanent MCAO model. Determination of the efficacy of the protection was carried out at different doses (see FIG. 3). As can be seen from FIG. 3, at least doses of 11 mg/kg, 3 mg/kg, 0.3 mg/kg and 0.03 mg/kg, contribute to a cerebral protection. The best protection is observed at the dose of 0.03 mg/kg.

[0134] FIG. 4 illustrates the evaluation of neuroprotection by an inventive chimeric peptide according to SEQ ID NO: 11 after i.v. administration against focal cerebral ischemia, in a transient MCAO model. Subsequent to provoking ischemia in adult mice, the mice were killed 48 h after reperfusion. Serial cryostat sections were prepared and infarct volumes were calculated. As can be seen from FIG. 4, the inventive chimeric peptide provides efficient neuroprotection.

[0135] FIG. 5 shows the results of an assay on neuronal cultures carried out by measuring LDH release following NMDA stimulation. The results clearly indicate a neuroprotective effect of the inventive chimeric D-JNKI1 peptide (SEQ ID NO: 11), since degenerative changes due to NMDA exposure were completely inhibited as indicated by the absence of significant LDH release above controls.

[0136] FIG. 6 depicts the results of the inhibition of endogeneous JNK-activity in HepG2 cells using inventive fusion peptides according to SEQ ID NOs: 9 and 11 in an one-well approach. As can be seen from FIG. 6, particularly panel d in FIG. 6, D-TAT-IB1(s) according to SEQ ID NO: 11 (here abbreviated as D-JNKI) effectively inhibits JNK activity, even better than L-TAT-IB1(s) according to SEQ ID NO: 9 (here abbreviated as L-JNKI).

[0137] FIG. 7 shows the protecting effect of D-TAT-IB1(s) Protection against permanent hearing loss. Changes of the hearing threshold level (dB sound pressure level) in guinea pigs following noise trauma (120 dB at 6 kHz during 30 minutes) at 8 kHz, the maximally impacted frequency, measured 20 minutes (temporary threshold shift, TTS, grey) and 15 days post noise exposure (permanent threshold shift). Guinea pigs received D-TAT-IB1(s) in a hyaluronic acid gel deposited onto the cochlear round window membrane either 30 minutes before, 30 minutes after or 4 hours after noise trauma; untreated ears served as control. TTS was measured 20 minutes post noise trauma, while PTS (black), which corresponds to permanent hearing loss, was determined after 15 days. As shown, D-TAT-IB1(s) not only protects substantially against permanent hearing loss from noise trauma if applied preventively before the noise exposure, but also in a time-dependent fashion if administered after trauma. PTS in treated ears was significantly lower for administration of D-TAT-IB1(s) 30 minutes and 4 hours post trauma than in untreated control ears.

EXAMPLES

Example 1: Identification of JNK Inhibitor Sequences

[0138] Amino acid sequences important for efficient interaction with JNK were identified by sequence alignments between known JBDs. A sequence comparison between the JBDs of IB1 [SEQ ID NO: 13], IB2 [SEQ ID NO: 14], c-Jun [SEQ ID NO: 15] and ATF2 [SEQ ID NO: 16] 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.

[0139] These two blocks are contained within a peptide sequence of 19 amino acids in L-IB1(s) [SEQ ID NO: 1] and are also shown for comparative reasons in a 23 aa peptide sequence derived from IB1 [SEQ ID NO: 17]. These sequences are shown in FIG. 1B, dashes in the L-IB1 sequence indicate a gap in the sequence in order to align the conserved residues with L-IB1(s).

Example 2: Preparation of JNK Inhibitor Fusion Proteins

[0140] Inventive JNK inhibitor fusion proteins according to SEQ ID NO: 9 were synthesized by covalently linking the C-terminal end of SEQ ID NO: 1 to a N-terminal 10 amino acid long carrier peptide derived from the HIV-TAT4g 57 (Vives et al., J Biol. Chem. 272: 16010 (1997)) according to SEQ ID NO: 5 via a linker consisting of two proline residues. This linker was used to allow for maximal flexibility and prevent unwanted secondary structural changes. The basic constructs were also prepared and designated L-IB1(s) (SEQ ID NO: 1) and L-TAT [SEQ ID NO: 5], respectively.

[0141] All-D retro-inverso peptides according to SEQ ID NO: 11 were synthesized accordingly. The basic constructs were also prepared and designated D-IB1(s) [SEQ ID NO: 2] and D-TAT [SEQ ID NO: 6], respectively.

[0142] All inventive D and L fusion peptides according to SEQ ID NOs: 9, 10, 11 and 12 were produced by classical Fmock synthesis and further analysed by Mass Spectrometry.

[0143] They were finally purified by HPLC. To determine the effects of the proline linker, 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. Generic peptides showing the conserved amino acid residues are given in FIG. 2.

Example 3: Inhibition of Cell Death by JBD19

[0144] Effects of the 19 aa long JBD sequence of IB1(s) on JNK biological activities were studied. The 19 aa sequence was linked N-terminal to the Green Fluorescent Protein (GFP JBD19 construct), and the effect of this construct on pancreatic β-cell apoptosis induced by IL1 was evaluated. 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).

[0145] Oligonucleotides corresponding to JBD19 and comprising a conserved sequence of 19 amino acids as well as a sequence mutated at the fully conserved regions 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 βTC-3 cells were cultured in RPMI 1640 medium supplemented with 10% Fetal Calf Serum, 100 μg/mL Streptomycin, 100 units/mL Penicillin and 2 mM Glutamine. Insulin producing OTC-3 cells were transfected with the indicated vectors and IL-1β (10 ng/mL) was added to the cell culture medium. The number of apoptotic cells was counted at 48 hours after the addition of IL-1β using an inverted fluorescence microscope. Apoptotic cells were discriminated from normal cells by the characteristic “blebbing out” of the cytoplasm and were counted after two days.

[0146] GFP is Green Fluorescent protein expression vector used as a control; JBD19 is the vector expressing a chimeric GFP linked to the 19 aa sequence derived from the JBD of IB1; JBD19Mut is the same vector as GFP-JBD19, but with a JBD mutated at four conserved residues shown as FIG. 1B; and JBD.sub.1-280 is the GFP vector linked to the entire JBD (aa 1-280). The GFP-JBD19 expressing construct prevented IL-1 β induced pancreatic β-cell apoptosis as efficiently as the entire JBD.sub.1-280.

[0147] As additional controls, sequences mutated at fully conserved IB1(s) residues had greatly decreased ability to prevent apoptosis.

Example 4: Cellular Import of TAT-IB1(s) Peptides

[0148] The ability of the L- and D-enantiomeric forms of TAT and inventive TAT-IB1(s) peptides (“TAT-IB peptides”) to enter cells was evaluated. L-TAT, D-TAT, inventive L-TAT-IB1(s), and inventive D-TAT-IB1(s) peptides [SEQ ID NOs: 5, 6, 9 and 12, respectively] were labeled by N-terminal addition of a glycine residue conjugated to fluorescein. Labeled peptides (1 μM) were added to βTC-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 μM, 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 μM BSA, 12 moles fluorescein/mole BSA) did not enter the cells.

[0149] 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 inventive D-TAT-IB1(s) were extremely stable inside the cells.

[0150] Fluorescent signals from these all-D retro-inverso peptides were still very strong 1 week later, and the signal was only slightly diminished at 2 weeks post treatment.

Example 5: In Vitro Inhibition of c-JUN, ATF2 and Elk1 Phosphorylation

[0151] The effects of the peptides on JNKs-mediated phosphorylation of their target transcription factors were investigated in vitro. Recombinant and non activated 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 inventive L-TAT or L-TAT-IB1(s) peptides (0-25 μM) 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 pCi .sup.33P-γ-dATP and 1 μg of either GST-Jun (aa 1-89), GST-AFT2 (aa 1-96) or GST-ELK1 (aa 307-428). GST-fusion proteins were purchased from Stratagene (La Jolla, Calif.).

[0152] Ten μL 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 inventive TAT-IB(s) peptide doses as low as 2.5 μM. However, a marked exception was the absence of TAT-IB(s) inhibition of JNK3 phosphorylation of Elk1. Overall, the inventive TAT-IB1(s) peptide showed superior effects in inhibiting JNK family phosphorylation of their target transcription factors. The ability of D-TAT, inventive D-TAT-IB1(s) and inventive L-TAT-IB1(s) peptides (0-250 μM dosage study) to inhibit GST-Jun (aa 1-73) phosphorylation by recombinant JNK1, JNK2, and JNK3 by were analyzed as described above. Overall, D-TAT-IB1(s) peptide decreased JNK-mediated phosphorylation of c-Jun, but at levels approximately 10-20 fold less efficiently than L-TAT-IB1(s).

Example 6: Inhibition of c-JUN Phosphorylation by Activated JNKs

[0153] The effects of the L-TAT or inventive L-TAT-IB1(s) 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 β treated PTC cells. PTC cells were cultured as described above. HeLa cells were cultured in DMEM medium supplemented with 10% Fetal Calf Serum, 100 μg/mL Streptomycin, 100 units/ml Penicillin and 2 mM Glutamine. One hour prior to being used for cell extract preparation, PTC cells were activated with IL-1 β 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-1β treated βTC-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 mMP-glycerophosphate, 1 mM dithiothreitol). Debris was removed by centrifugation for five minutes at 15,000 rpm in an SS-34 Beckman rotor. One-hundred μg extracts were incubated for one hour at room temperature with one μg GST-jun (amino acids 1-89) and 10 μL of glutathione-agarose beads (Sigma). Following four washes with the scraping buffer, the beads were resuspended in the same buffer supplemented with L-TAT or inventive L-TAT-IB1(s) peptides (25 μM) for 20 minutes. Kinase reactions were then initiated by addition of 10 mM MgCl.sub.2 and 5 pCi .sup.33P-γ-dATP and incubated for 30 minutes at 30° C.

[0154] 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).

[0155] The inventive TAT-IB(s) peptides efficiently prevented phosphorylation of c-Jun by activated JNKs in these experiments.

Example 7: In Vivo Inhibition of c-JUN Phosphorylation by Inventive TAT-IB(s) Peptides

[0156] To determine whether the inventive 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 5×GAL-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, 3×10.sup.5 cells were transfected with the plasmids in 3.5-cm dishes using DOTAP (Boehringer Mannheim) following instructions from the manufacturer. For experiments involving GAL-Jun, 20 ng of the plasmid was transfected with 1 μg of the reporter plasmid pFR-Luc (Stratagene) and 0.5 μg of either MKK4 or MKK7 expressing plasmids. Three hours following transfection, cell media were changed and TAT and TAT-IB1(s) peptides (1 M) were added. The luciferase activities were measured 16 hours later using the “Dual Reporter System” from Promega after normalization to protein content. Addition of TAT-IB1(s) peptide blocked activation of c-Jun following MKK4 and MKK7 mediated activation of JNK. Because HeLa cells express JNK1 and JNK2 isoforms but not JNK3, we transfected cells with JNK3. Again, the TAT-IB(s) peptide inhibited JNK2 mediated activation of c-Jun.

Example 8: Inhibition of IL-1 Induced Pancreatic B-Cell Death by TAT-IB Peptides

[0157] We investigated the effects of the inventive L-TAT-IB(s) peptides on the promotion of β-cell apoptosis elicited by IL-1. βTC-3 cell cultures were incubated for 30 minutes with 1 μM of inventive L-TAT-IB1(s) peptides followed by 10 ng/mL of IL-1. A second addition of peptide (1 μM) was performed 24 hours later. Apoptotic cells were counted after two days of incubation with IL-1 β using propidium iodide (red stained cell are dead cells) and Hoechst 33342 (blue stained cell are cells with intact plasma membrane) nuclear staining. Addition of the inventive TAT-IB(s) peptides inhibited IL-1-induced apoptosis of βTC-3 cells cultured in the presence of IL-1 β for two days.

[0158] Long term inhibition of IL-1 induced cells death was examined by treating βTC-3 cells as described above, except that incubation of the cells with the peptides and IL-1 β was sustained for 12 days. Additional peptides (1 μM) were added each day and additional IL-1 β (10 ng/mL) was added every 2 days. The inventive TAT-IB1(s) peptide confers strong protection against apoptosis in these conditions. Taken together, these experiments provide evidence that inventive TAT-IB(s) peptides are biologically active molecules able to prevent the effects of JNK signaling on cell fate.

Example 9: Synthesis of Inventive all-D Retro-Inverso IB(s) Peptides

[0159] 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.

[0160] 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: 5], the retro-inverso peptide analog of this peptide (formed of D-amino acids) would have the sequence RRRQRRKKRG [SEQ ID NO: 6]. 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 analyzed by Mass Spectrometry. They were finally purified by HPLC.

[0161] 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 Inventive all-D Retro-Inverso IB(s) Peptides

[0162] Long term biological activity is predicted for the inventive D-TAT-IB(s) retro-inverso containing peptide heteroconjugate when compared to the native L-amino acid analog owing to protection of the inventive D-TAT-IB(s) peptide from degradation by native proteases, as shown in Example 5.

[0163] Inhibition of IL-1 β induced pancreatic β-cell death by the inventive D-TAT-IB1(s) peptide was analyzed. βTC-3 cells were incubated as described above for 30 minutes with one single addition of the indicated peptides (1, μM), then IL-1 (10 ng/ml) was added.

[0164] Apoptotic cells were then counted after two days of incubation with IL-1 β 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.

[0165] Long term inhibition of IL-IP induced cell-death by the D-TAT-IB1 peptide was also analyzed. βTC-3 cells were incubated as above for 30 minutes with one single addition of the indicated peptides (1 μM), then IL-1 β (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 by use of propidium iodide and Hoechst 33342 nuclear staining. Note that one single addition of the TAT-IB1 peptide does not confer long-term protection. A minimum of 1.000 cells were counted for each experiment. As a result, inventive D-TAT-IB1(s), but not inventive L-TAT-IB1(s), was able to confer long term (15 day) protection.

Example 11: Inhibition of Irradiation Induced Pancreatic B-Cell Death by TAT-IB(s) Peptides

[0166] JNK is also activated by ionizing radiation. To determine whether inventive TAT-IB(s) peptides would provide protection against radiation-induced JNK damage, “WiDr” cells were irradiated (30 Gy) in presence or absence of D-TAT, inventive L-TAT-IB1(s) or inventive D-TAT-IB1(s) peptides (1 μM added 30 minutes before irradiation). Control cells (CTRL) were not irradiated. Cells were analyzed 48 hours later by means of PI and Hoechst 3342 staining, as described above. N=3, SEM are indicated. Inventive L-TAT-IB1(s) and D-TAT-IB1(s) peptides were both able to prevent irradiation induced apoptosis in this human colon cancer line.

Example 12: Radioprotection to Ionizing Radiation by Inventive TAT-IB(s) Peptides

[0167] To determine the radioprotective effects of the inventive TAT-IB(s) peptides, C57B1/6 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 TAT, inventive L-TAT-IB1(s) or inventive D-TAT-IB1(s) peptides (301 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.

[0168] 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.

[0169] 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.

[0170] The results of these experiments indicate that the inventive TAT-IB(s) peptides can protect against weight loss and erythema/edema associated with ionizing radiation.

Example 13: Suppression of JNK Transcription Factors by Inventive L-TAT-IB1(s) Peptides

[0171] Gel retardation assays were carried out with an AP-1 doubled labeled probe (5′-CGC TTG ATG AGT CAG CCG GAA-3′ (SEQ ID NO: 27). HeLa cell nuclear extracts that were treated or not for one hour with 5 ng/mlTNF-α, as indicated. TAT and inventive L-TAT-IB1(s) peptides were added so minutes before TNF-α. 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.

[0172] Inventive L-TAT-IB1(s) peptides decrease the formation of the AP-1 DNA binding complex in the presence of TNF-α.

Example 14: Evaluation of the Neuroprotection Against Focal Cerebral Ischemia, in a Permanent MCAO Model—Determination of the Efficacy of the Protection at Different Doses. (See FIG. 3)

[0173] Focal cerebral ischemia was induced in 12-days-old rats. Pups were anesthetized in an induction chamber with 2% isoflurane and during the operation anaesthesia was maintained using a mask under 2% isoflurane. MCAO was induced by electrocoagulating a main branch of the middle cerebral artery (MCA). Rats were placed on the right side, and an oblique dermal incision was made between the ear and eye. After excision of the temporal muscle, the cranial bone was removed from the frontal suture to a level below the zygomatic arch. The left MCA, exposed just after its apparition over the rhinal fissure, was permanently electrocoagulated at the inferior cerebral vein level before the MCA bifurcated into frontal and parietal branches. The cranial skin incision was then closed. Rat pups were then placed in an incubator maintained at 37° C. until they awoke, and were then transferred to their mother. 6 hours later an inventive chimeric D-TAT-IB1(s) peptide according to SEQ ID NO: 11 was injected intraperitoneally. 24 hours after the coagulation, the rats were anesthetized with chloral hydrate and perfused through the ascending aorta with 4% paraformaldehyde in PBS. Brains were then removed and kept for 2 hours in the same fixative solution, and placed in a gradient of 30% sucrose in PBS for about 15 hours at 4° C. Brains were frozen in isopentane (−40° C.) and stored at −20° C. Coronal cryostat sections of 50 □m were collected on glass slides. The sections were stained with cresyl violet. Each tenth section was analyzed and the total volume of the lesion was calculated using the Neuroleucida programme. In the control group A, the mean lesion volume was 21.47 mm.sup.3. All the treated groups have a lower mean than the control group. A significant statistic difference is observed between group A and groups C, E and F (one-tailed t-test, p=0.030, p=0.002, p=0.001 respectively). The results are shown in FIG. 4.

[0174] As a result, these data support the conclusion that the inventive chimeric D-TAT-IB1(s) peptide according to SEQ ID NO: 11, administered at a dose of 11 mg/kg, 3 mg/kg, 0.3 mg/kg and 0.03 mg/kg, contributes to a cerebral protection. Results at a dose of 1 mg/kg, 0.003 mg/kg and 0.0003 compared to saline group suggest that the total sample was not large enough to reach a significant difference. The best protection is observed at the dose of 0.03 mg/kg.

Example 15: Evaluation of Neuroprotection by Inventive Chimeric Peptides after IV Administration Against Focal Cerebral Ischaemia, in a Transient MCAO Model (See FIG. 4)

Transient Ischemia in Adult Mice.

[0175] Using male ICR-CD1 mice (6 weeks old; 18-37 g; Harlan), we provoked ischemia by introducing a filament from the common carotid artery into the internal carotid and advancing it into the arterial circle, thereby occluding the middle cerebral artery. We measured regional cerebral blood flow by laser Doppler flowmetry, with a probe fixed on the skull throughout the ischemia until 10 min after reperfusion. Rectal temperature was measured and maintained at 37° C. The mice were killed 48 h after reperfusion. Serial cryostat sections 20 m thick were traced using a computer-microscope system equipped with the Neurolucida program (MicroBrightField) and the volumes of the ischemic area and of the whole brain were calculated (blinded) with the Neuroexplorer program.

[0176] XG-102 0.3=0.3 mg/kg, XG-102 1=1 mg/kg, XG-102 5=5 mg/kg

[0177] The infarct volume sizes (mm.sup.3) after bolus iv administration of placebo and XG-102 0.3, 1.3 mg/kg 6 hours after reperfusion (30 minutes clamp) in an adult mice model were as follows.

TABLE-US-00003 infarcts moyenne écart type control n = 5 72 17 XG102 0.3 n = 5 16 4 XG102 1 n = 1 16 XG102 3 n = 5 15 5

Example 16: Assay on Neuronal Cultures by Measuring LDH Release Following NMDA Stimulation (See FIG. 5)

[0178] The neuroprotective effect of the D-TAT-IB (generic)(s)/D-JNKI1 peptide (SEQ ID NO: 12) was evaluated in sister cultures pre-treated for 30 min with the indicated concentrations of peptides or MK-801 before continuous exposure to 100 μM NMDA. After 12 h of NMDA treatment, in cultures pretreated with 5 μM of D-TAT-IB (generic)(s)/D-JNKI1 the degenerative changes due to NMDA exposure were completely inhibited as indicated by the absence of significant LDH release above controls (FIG. 5). The morphological appearance, number and distribution of the neurons were indistinguishable from the controls.

Cortical Neuronal Culture.

[0179] We dissected small pieces of cortex from the brains of two day old rat pups, incubated them with 200 units of papain for 30 min at 34° C., and then plated the neurons at densities of approximately 1×10.sup.6 cells/plate on dishes pre-coated with 100 μg/ml poly-D-lysine. The plating medium consisted of B27/Neurobasal (Life Technologies, Gaithersburg, Md.) supplemented with 0.5 mM glutamine, 100 U/ml penicillin and 100 ug/ml streptomycin.

Lactate Dehydrogenate (LDH) Cytotoxicity Assay.

[0180] LDH released into the bathing medium 12, 24 and 48 h after NMDA administration was measured using the Cytotox 96 non-radioactive cytotoxicity assay kit (Promega, WI) (see FIG. 5).

Example 17: Inhibition of Endogenous JNK Activity in HepG2 Cells Using an all-in One Well Approach (See FIG. 6)

[0181] HepG2 cells were seeded at 3'000 cells/well the day prior the experiment. Then, increasing concentrations of either interleukin-1β [IL-1β((ν)] or tumor necrosis factor α [TNFα(.square-solid.)] (a) were added to activate JNK for 30 minutes. Cells were lysed in 20 mM Hepes, 0.5% Tween pH 7.4 and processed for AlphaScreen JNK. (b) Z′ for the JNK activity induced by 10 ng/ml IL-1β and measured in 384 wells/plate (n=96). (c) Inhibition of endogenous IL-1 β-induced JNK activity with chemical JNK inhibitors [staurosporin (°) and SP600125 (.square-solid.)]. (d) Effect of peptidic inhibitors L-TAT-IB1(s) according to SEQ ID NO: 9 [here abbreviated as L-JNKi (ν)) and D-TAT-IB1(s) according to SEQ ID NO: 11 (here abbreviated as D-JNKi (.diamond-solid.)) and JBDs (.square-solid.) (corresponds to L-JNKI without the TAT sequence)] on IL-1α dependent JNK activity. All panels are representative of three independent experiments (n=3).

Methods: Alphascreen Kinase Assay

Principle:

[0182] AlphaScreen is a non-radioactive bead-based technology used to study biomolecular interactions in a microplate format. The acronym ALPHA stands for Amplified Luminescence Proximity Homogenous Assay. It involves a biological interaction that brings a “donor” and an “acceptor” beads in close proximity, then a cascade of chemical reactions acts to produce an amplified signal. Upon laser excitation at 680 nm, a photosensitizer (phthalocyanine) in the “donor” bead converts ambient oxygen to an excited singlet state. Within its 4 μsec half-life, the singlet oxygen molecule can diffuse up to approximately 200 nm in solution and if an acceptor bead is within that proximity, the singlet oxygen reacts with a thioxene derivative in the “acceptor” bead, generating chemiluminescence at 370 nm that further activates fluorophores contained in the same “acceptor” bead. The excited fluorophores subsequently emit light at 520-620 nm. In the absence of an acceptor bead, singlet oxygen falls to ground state and no signal is produced.

[0183] Kinase reagents (B-GST-cJun, anti P-cJun antibody and active JNK3) were first diluted in kinase buffer (20 mM Tris-HCl pH 7.6, 10 mM MgCl.sub.2, 1 mM DTT, 100 μM Na.sub.3VO.sub.4, 0.01% Tween-20) and added to wells (15 μl). Reactions were then incubated in presence of 10 μM of ATP for 1 h at 23° C. Detection was performed by an addition of 10 μl of beads mix (Protein A acceptor 20 μg/ml and Streptavidin donor 20 μg/ml), diluted in detection buffer (20 mM Tris-HCl pH 7.4, 20 mM NaCl, 80 mM EDTA, 0.3% BSA), followed by an another one-hour incubation at 23° C. in the dark. For measurement of JNK endogenous activity, kinase assays were performed as described above except active JNK3 was replaced by cells lysates and reaction kinase components were added after the cells lysis. B-GST-cjun and P-cJun antibody were used at the same concentrations whereas ATP was used at 50 μM instead of 10 μM. AlphaScreen signal was analyzed directly on the Fusion or En Vision apparatus.

Example 18: Treatment of Noise Trauma

[0184] D-TAT-IB1(s) was applied onto the round window membrane of the cochlea of 3 groups of guinea pigs (each group with 6 animals) in 2 microliters of a gel formulation of 2.6% buffered hyaluronic acid (Hylumed, Genzyme Corp.) at a concentration of 100 □M either 30 minutes before noise trauma (120 dB at 6 kHz during 30 minutes) or 30 minutes or 4 hours thereafter. Untreated ears served as control. Hearing threshold shifts were evaluated by auditory brainstem response measurements 20 minutes after noise trauma (temporary threshold shift, TTS) and 15 days following the trauma (permanent threshold shift, PTS). Administration of D-TAT-IB1(s) protected against permanent hearing loss even if applied after exposure to excessive noise compared to non-treated ears. The protective effect was stronger the earlier D-TAT-IB1(s) was administered after the noise trauma. Thus, D-TAT-IB1(s) is a very effective otoprotective compound in case of noise trauma.

[0185] From the foregoing detailed description of the specific embodiments of the invention, it should be apparent that unique cell-permeable bioactive chimeric peptides and JNK inhibitor sequences 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.