Inhibitors of NF-&kgr;B Activation

Details Inhibitors of NF-&kgr;B Activation Inhibitors of NF-&kgr;B Activation

2000-10-31

2004-01-06

Low, Christopher S. F. (Department: 1653)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

C514S002600, C930S010000

Reexamination Certificate

active

06673897

ABSTRACT:

TECHNICAL FIELD
This invention relates to novel inhibitors of the Nuclear factor kappa B (NF-&kgr;B) activating pathway useful in the treatment of NF-&kgr;B related diseases and/or in the improvement of anti-tumor treatments. The invention also relates to nucleic acids coding for the novel inhibitors. The invention relates further to the use of polypeptides, derived from these inhibitors in the treatment of NF-&kgr;B related diseases and/or cancer. Furthermore, the invention concerns pharmaceutical preparations, comprising the novel inhibitors or the polypeptides, derived from these inhibitors.
BACKGROUND
NF-&kgr;B is an ubiquitously expressed transcription factor that controls the expression of a diverse range of genes involved in inflammation, immune response, lymphoid differentiation, growth control and development. NF-&kgr;B resides in the cytoplasm as an inactive dimer consisting of p50 and p65 subunits, bound to an inhibitory protein known as I&kgr;B. The latter becomes phosphorylated and degraded in response to various environmental stimuli, such as pro-inflammatory cytokines, viruses, lipopolysaccharides, oxidants, UV light and ionizing radiation. This allows NF-&kgr;B to translocate to the nucleus where it activates genes that play a key role in the regulation of inflammatory and immune responses, including genes that encode pro-inflammatory cytokines (IL-1&bgr;, TNF, GM-CSF, IL-2, IL-6, IL-11, IL-17), chemokines (IL-8, RANTES, MIP-1&agr;, MCP-2), enzymes that generate mediators of inflammation (NO synthetase, cyclo-oxygenase), immune receptors (IL-2 receptor) and adhesion molecules (ICAM-1, VCAM-1, E-selectin). Some of these induced proteins can on their turn activate NF-&kgr;B, leading to the further amplification and perpetuation of the inflammatory response. Recently, NF-&kgr;B has been shown to have an anti-apoptotic role in certain cell types, most likely by inducing the expression of anti-apoptotic genes. This function may protect tumor cells against anti-cancer treatments and opens the possibility to use NF-&kgr;B inhibiting compounds to sensitize the tumor cells and to improve the efficiency of the anti-cancer treatment.
Because of its direct role in regulating responses to inflammatory cytokines and endotoxin, activation of NF-&kgr;B plays an important role in the development of different diseases such as (Barnes and Karin, 1997): chronic inflammatory diseases, i.e., rheumatoid arthritis, asthma and inflammatory bowel disease (Brand et al., 1996); acute diseases, i.e., septic shock (Remick, 1995); Alzheimer's disease where the &bgr;-amyloid protein activates NF-&kgr;B (Behl et al., 1997); atherosclerosis, where NF-&kgr;B may be activated by oxidized lipids (Brand et al., 1997); autoimmune diseases, i.e., such as systemic lupus erythematosis (Kaltschmidt et al., 1994); or cancer by up-regulating certain oncogenes or by preventing apoptosis (Luque et al., 1997). In addition, NF-&kgr;B is also involved in viral infection since it is activated by different viral proteins, such as occurs upon infection with rhinovirus, influenza virus, Epstein-Barr virus, HTLV, cytomegalovirus or adenovirus. Furthermore, several viruses such as HIV have NF-&kgr;B binding sites in their promoter/enhancer regions (Mosialos, 1997).
Because of the potential role of NF-&kgr;B in many of the above mentioned diseases, NF-&kgr;B and its regulators have drawn much interest as targets for the treatment of NF-&kgr;B related diseases. Glucocorticoids are effective inhibitors of NF-&kgr;B, but they have endocrine and metabolic side effects when given systematically (Barnes et al., 1993). Antioxidants may represent another class of NF-&kgr;B inhibitors, but currently available antioxidants, such as acetyl-cysteine are relatively weak and unspecific (Schreck et al., 1991). Aspirin and sodium salicylate also inhibit activation of NF-&kgr;B, but only at relatively high concentrations (Kopp and Gosh, 1994). There are some natural inhibitors of NF-&kgr;B such as glyotoxin, derived from Aspergillus, but these compounds are too toxic to be used as a drug (Pahl et al., 1996). Finally, there may be endogenous inhibitors of NF-&kgr;B, such as IL-10, that blocks NF-&kgr;B through an effect on I&kgr;B (Wang et al., 1995). However, total inhibition of NF-&kgr;B in all cell types for prolonged periods is unwanted, because NF-&kgr;B plays a crucial role in the immune response and other defensive responses.
An important role in the induction of NF-&kgr;B by TNF and IL1 has recently been demonstrated for TNF-receptor associated factors, TRAF2 and TRAF6, which are recruited to the stimulated TNF-receptor and IL-1 receptor, respectively (Rothe et al., 1995; Cao et al., 1996). Over expression of TRAF2 or TRAF6 activates NF-&kgr;B, whereas dominant negative mutants inhibit TNF or IL-1 induced activation of NF-&kgr;B in most cell types. TRAF2 knock out studies have recently shown that TRAF2 is not absolutely required for NF-&kgr;B activation, presumably because of redundancy within the TRAF family (Yeh et al. 1997). The TRAF induced signaling pathway to NF-&kgr;B was further resolved by the identification of the TRAF-interacting protein NIK, which mediates NF-&kgr;B activation upon TNF and IL-1 stimulation by association and activation of I&kgr;B kinase-&agr; and -&bgr; (IKK) (Malinin et al, 1997; Regnier et al., 1997; DiDonato et al., 1997; Zandi et al., 1997; Woronicz et al., 1997). The latter are part of a large multi-protein NF-&kgr;B activation complex and are responsible for phosphorylation of I&kgr;B, leading to its subsequent degradation and to translocation of released, active NF-&kgr;B to the nucleus. This allows a more specific inhibition of NF-&kgr;B activation by stimuli (including TNF and IL-1) that activate TRAF pathways. Based on this principle, WO 97/37016 discloses the use of NIK and other TRAF interacting proteins for the modulation of NF-&kgr;B activity.
Another protein that can associate with TRAF2 is the zinc finger protein A20 (Song et al., 1996). The latter is encoded by an immediate early response gene induced in different cell lines upon stimulation by TNF or IL-1 (Dixit et al, 1990). Interestingly, over expression of A20 blocks both TNF and IL-1 induced NF-&kgr;B activation (Jaattela et al., 1996). However, the mechanism by which A20 blocks NF-&kgr;B activation is totally unknown. In contrast to NIK, A20 does not seem to act directly on I&kgr;B resulting in alternative pathway to modulate NF-&kgr;B activation.
De Valck et al. (1997) isolated an A20 binding protein, so-called 14-3-3, using a yeast two-hybrid assay and demonstrated that NF-&kgr;B inhibition was independent from the binding of A20 to 14-3-3.
DESCRIPTION OF THE INVENTION
It is shown herein that other new A20 interacting proteins unexpectedly can modulate and/or inhibit NF-&kgr;B activation.
The invention includes an isolated functional protein comprising an amino acid sequence with 70-100% homology to the amino acid sequence depicted in SEQ. ID. NO. 2, or comprising an amino acid sequence with 70-100% homology to the amino acid sequence depicted in SEQ. ID. NO. 3, or, in the alternative, comprising an amino acid sequence with 70-100% homology to the amino acid sequence depicted in SEQ. ID. NO. 5.
More specifically, the functional protein comprises an amino acid sequence with 70-100% homology to the amino acids 54-647 of SEQ. ID. NO. 2, even more specifically the functional protein comprises an amino acid sequence with 70-100% homology to the amino acids 390-647 of SEQ. ID. NO. 2, or, in the alternative, and/or comprising an amino acid sequence with 70-100% homology to the amino acids 420-647 of SEQ. ID. NO.2.
Homology, in this context, means identical or similar to the referenced sequence, while obvious replacements/modifications of any of the amino acids provided, are included as well. A homology search in this respect can be performed with the BLAST-P (Basic Local Alignment Search Tool) program well known to a person skilled in the art. For the corresponding nucleic acid sequence homology is referred to the BLASTX and

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