Cell-permeable peptide inhibitors of the JNK signal transduction pathway

An inhibitor, JNK technology, applied in the field of inhibitors of protein kinase c-Jun amino-terminal kinase and pharmaceutical compounds with pathophysiological abnormalities, can solve the problem of high cost of inhibitor sequence separation and purification steps

Active Publication Date: 2008-09-10
希根因弗兰曼斯有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Furthermore, according to WO 01 / 27268, isolation and purification steps of inhibitor sequences are costly, especially for the preparation of huge quantities (e.g. for industrial production)

Method used

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  • Cell-permeable peptide inhibitors of the JNK signal transduction pathway
  • Cell-permeable peptide inhibitors of the JNK signal transduction pathway
  • Cell-permeable peptide inhibitors of the JNK signal transduction pathway

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0258] Example 1: Identification of JNK Inhibitor Sequences

[0259] Amino acid sequences important for effective interaction with JNK were identified by sequence alignment among known JBDs. 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] defines weak retention The 8 amino acid sequence of ( figure 1 A). Because the JBDs of IB1 and IB2 have approximately 100 folds, they are as efficient as binding c-Jun or ATF2 in JNK (Dickens et al. Science 277:693 (1997), which elaborates on the remaining residues between IB1 and IB2 The importance of bases for conferring maximum binding. A comparison between the JBDs of IB1 and IB2 defined two blocks of 7 amino acids and 3 amino acids that were highly conserved between these two sequences.

[0260] These two blocks are located in the 19 amino acid peptide sequence in L-IB1 [SEQ ID NO: 1] and are shown in the 23 aa peptide sequence derived from IB1 [SEQ ID ...

example 2

[0261] Example 2: Preparation of JNK inhibitor fusion protein

[0262] The JNK inhibitor fusion protein of the present invention according to SEQ ID NO: 9 can be synthesized by covalently linking the C-terminal of SEQ ID NO: 1 to the N-terminal of a 10 amino acid long carrier peptide, wherein the carrier peptide Derived from HIV-TAT4g 57 according to SEQ ID NO: 5 (Vives et al., J Biol. Chem. 272: 16010 (1997)), the linkage is via a linker consisting of two proline residues. This linker allows maximum flexibility and prevents undesired secondary structure changes. These two basic structures were prepared and assigned for L-IB1(s) (SEQ ID NO: 1) and L-TAT [SEQ ID NO: 5], respectively.

[0263] Thus, an all-D retro-inverso peptide according to SEQ ID NO: 11 can be synthesized. These two basic structures were also prepared and assigned from D-IB1 [SEQ ID NO: 2] and D-TAT [SEQ ID NO: 6], respectively.

[0264] D and L fusion peptides of the present invention according to SEQ I...

example 3

[0265] Example 3: Inhibition of cell death by JBD19

[0266] The role of the 19 aa long JBD on JNK biological activity was studied. The 19aa sequence is linked to the N-terminus of green fluorescent protein (GFP JBD19 construct), and it is estimated that this structure is related to IL1-induced apoptosis of pancreatic cells. Apoptotic patterns were previously shown and masked by transfection with JBD1-280, however, specific inhibitors of ERK1 / 2 or p38 did not confer protection (eg Ammendrup et al., supra).

[0267] Synthesis of an oligonucleotide corresponding to the JBD and having a conserved sequence of 19 amino acids, as well as a sequence that varies in the fully conserved region, and directly inserting the oligonucleotide and sequence into the coding green fluorescent protein (GFP, derived from cloning) EcoRI and SalI positions of pEGFP-N1. Insulin producing TC-3 cells was cultured in RPMI1640 medium, which also had 10% Fetal Calf Serum, 100 μg / mL streptomycin, 100 un...

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Abstract

The present invention refers to protein kinase inhibitors and more specifically to inhibitors of the protein kinase c-Jun amino terminal kinase. Additionally, the present invention provides JNK inhibitor sequences, chimeric peptides, nucleic acids encoding same as well as pharmaceutical compositions for treating pathophysiologies associated with JNK signaling.

Description

technical field [0001] The present invention relates to protein kinase inhibitors, more particularly, to inhibitors of protein kinase c-Jun N-terminal kinase (c-Jun N-terminal kinase, JNK). In addition, the present invention provides JNK inhibitor sequences, chimeric peptides, nucleic acids encoding the same, and pharmaceutical compositions useful for treating pathophysiological abnormalities associated with JNK signaling. Background technique [0002] N-terminal kinase is one of the components of the stress-activated group of mitogen-activated protein (MAP) kinases. These kinases are used to control the growth and differentiation of cells and, more generally, to respond to the cell's environmental stimuli. The JNK signaling pathway is activated in response to environmental stress by binding several classes of cell surface receptors. These receptors include cytokine receptors, helix receptors and receptor tyrosine kinases. In mammalian cells, JNK is used in biological pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47
CPCC07K7/08C07K14/4703C07K14/47C07K14/005A61K38/00A61P11/00A61P11/16A61P17/00A61P17/02A61P17/06A61P19/02A61P27/16A61P29/00A61P3/10A61P31/04A61P31/18A61P35/00A61P35/02A61P37/00A61P37/02A61P37/04A61P37/06A61P37/08A61P39/06A61P43/00A61P7/08A61P9/00A61P9/10A61P9/12A61P9/14A61K38/17C07K19/00C12N15/62C07K16/18C07K2319/10C12N7/00C12N2740/16322C12N2740/16371
Inventor 克里斯托夫·邦尼
Owner 希根因弗兰曼斯有限公司
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