Compounds and methods for modulating activation of NF-&kgr;B

Details Compounds and methods for modulating activation of NF-&kgr;B Compounds and methods for modulating activation of NF-&kgr;B

2001-04-09

2003-12-02

Slobodyansky, Elizabeth (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

C530S324000, C530S325000, C530S326000, C530S327000, C530S328000, C530S350000

Reexamination Certificate

active

06656713

ABSTRACT:

TECHNICAL FIELD
The present invention relates generally to compositions and methods for modulating the activation of nuclear factor &kgr;B (NF-&kgr;B). The invention is more particularly related to agents that modulate ubiquitination of phosphorylated I&kgr;B&agr; and/or I&kgr;B&bgr; and to methods for treating diseases associated with NF-&kgr;B activation. Modulating agents encompassed by the present invention include E3 ubiquitin ligases, and portions and variants thereof.
BACKGROUND OF THE INVENTION
NF-&kgr;B is a transcription factor that plays a pivotal role in the highly specific pattern of gene expression observed for immune, inflammatory and acute phase response genes, including interleukin 1, interleukin 8, tumor necrosis factor and certain cell adhesion molecules. Like other members of the Rel family of transcriptional activators NF-&kgr;B is sequestered in an inactive form in the cytoplasm of most cell types. A variety of extracellular stimuli including mitogens, cytokines, antigens, stress inducing agents, UV light and viral proteins initiate a signal transduction pathway that ultimately leads to NF-&kgr;B release and activation.
Important modulators of NF-&kgr;B activation are the inhibitor proteins I&kgr;B&agr; and I&kgr;B&bgr; (referred to herein as I&kgr;B), which associate with (and thereby inactivate) NF-&kgr;B in the cytoplasm of nonstimulated cells. Activation and nuclear translocation of NF-&kgr;B occurs following signal-induced phosphorylation of I&kgr;B, which leads to proteolysis via the ubiquitin pathway. For I&kgr;B&agr;, the stimulus-induced phosphorylation at serines 32 and 36 renders the inhibitor a target for ubiquitination at lysines 21 and 22, resulting in degradation. Similarly, phosphorylation of I&kgr;B&bgr; at serines 19 and 23 renders the inhibitor a target for ubiquitination at lysin 9. However. the component(s) of the ubiquitin system mediating I&kgr;B recognition have not been identified.
Degradation of a protein via the ubiquitin pathway proceeds by two discrete and successive steps: (a) covalent attachment of multiple ubiquitin molecules to the protein substrate, and (b) degradation of the targeted protein by the 26S proteasome complex. The ubiquitin pathway consists of several components that act in concert and in a hierarchical manner (for reviews, see Ciechanover,
Cell
79:13, 1994; Hochstrasser,
Curr. Op. Cell. Biol
. 7:215, 1995; Jentsch and Schlenker,
Cell
82:881, 1995; Deshaies,
Trends Cell Biol
. 5:428, 1995). One such component, a single E1 enzyme, carries out activation of ubiquitin. Several major species of E2 enzymes have been characterized in mammalian cells, plants, and yeast. E2 enzymes probably bind to the ligase E3 (Reiss and Hersko,
J. Biol. Chem
. 265:3685, 1990; Dohmen et al.,
Proc. Natl. Acad. Sci. USA
88:7351, 1991) and it appears that each E2 enzyme can act with one or more E3 proteins (Nuber et al.,
J. Biol. Chem
. 271:2795, 1996; Orian et al.,
J. Biol. Chem
. 270:21707, 1995; Stancovski et al.,
Mol. Cell. Biol
. 15:7106, 1995; Gonen et al.,
J. Biol. Chem
. 271:302, 1996).
Only few E3 enzymes (ubiquitin ligases) have been described. Mammalian E3&agr; (UBR1 in yeast) and E3&bgr; recognize protein substrates via their free N-terminal amino acid residues (“N-end rule”; Varshavsky,
Cell
69:725, 1992; Hershko and Ciechanover,
Ann. Rev. Biochem
. 61:761, 1992). Cdc53 is probably an E3 involved in targeting phosphorylated G1 cyclins (Willems et al.,
Cell
86:453, 1996). E6-AP is involved in recognition of p53 (Scheffner et al.,
Cell
75:495, 1993), and a series of unique E6-AP homologous proteins have been identified (Huibregtse et al.,
Proc. Natl. Acad. Sci. USA
92:2563, 1995): Nedd4 is involved the degradation of the epithelial Na
+
channel (Staub et al,
Embo J
. 15:2371, 1996) and RSP5 (NIP1) is involved in tagging the permeases Gap1 and Fur1 (Hein et al.,
Mol. Microbiol
. 18:77, 1995), whereas Pub1 targets Cdc25 (Nefsky and Beach,
EMBO J
. 15:1301, 1996). Several other E3 enzymes that have been recently isolated appear to be involved in the degradation of c-Fos, a subset of muscle proteins, and in the processing of p105, the NF-&kgr;B precursor (Orian et al.,
J. Biol. Chem
. 270:21707, 1995; Stancovski et al.,
Mol. Cell. Biol
. 15:7106, 1995; Gonen et al.,
J. Biol. Chem
. 271:302, 1996). Thus, it appears that the ligases represent a large, mostly unraveled family of enzymes and, except for the mode of recognition of the “N-end rule” ligases (E3&agr; and E3&bgr;), the recognition motifs of all other known substrates of the ubiquitin system have not been identified.
Accordingly, there is a need in the art for an improved understanding of I&kgr;B degradation via the ubiquitin pathway, and for the identification of modulators of this degradation process for use in treating diseases associated with activation of NF-&kgr;B. The present invention fulfills these needs and further provides other related advantages.
SUMMARY OF THE INVENTION
Briefly stated, the present invention provides compositions and methods for modulating the activation of nuclear factor &kgr;B (NF-&kgr;B) by modulating ubiquitination of phosphorylated I&kgr;B&agr; and/or I&kgr;B&bgr;. Within one aspect, the present invention provides isolated human E3 ubiquitin ligase polypeptides. Such polypeptides may comprise a human E3 ubiquitin ligase sequence as recited in SEQ ID NO:16, or a portion or variant thereof that differs in one or more amino acid substitutions, insertions, deletions and/or additions, such that the polypeptide (a) enhances ubiquitination of phosphorylated I&kgr;B or (b) binds to phosphorylated I&kgr;B and inhibits ubiquitination of phosphorylated I&kgr;B. Within certain embodiments, such a polypeptide may have the sequence recited in SEQ ID NO:16 or a variant thereof that differs in one or more amino acid deletions, insertions or substitutions at no more than 20% of the amino acid residues in SEQ ID NO:16, such that the polypeptide enhances ubiquitination of phosphorylated I&kgr;B. Within further embodiments, such a polypeptide may comprise a portion of a human E3 ubiquitin ligase, or variant of such a portion, wherein the portion binds to phosphorylated I&kgr;B and inhibits ubiquitination of phosphorylated I&kgr;B.
The present invention further provides, within other aspects, isolated polynucleotides that encode a polypeptide as described above. Within certain embodiments, such polynucleotides may encode a portion of a human E3 ubiquitin ligase, or variant of such a portion, as described above. Antisense polynucleotides comprising at least 10 consecutive nucleotides complementary to such a polynucleotide are also provided. Expression vectors comprising such a polynucleotide, and host cells transformed or transfected with such an expression vector, are further provided.
Within further aspects, the present invention provides pharmaceutical compositions comprising a polypeptide or polynucleotide as described above in combination with a physiologically acceptable carrier.
Within other aspects, the present invention provides isolated antibodies, and antigen binding fragments thereof, that bind to a human E3 ubiquitin ligase having a sequence recited in SEQ ID NO:16. Such antibodies may be monoclonal.
Within further aspects, pharmaceutical compositions are provided, comprising an antibody or fragment thereof as described above in combination with a physiologically acceptable carrier.
The present invention further provides methods for modulating NF-&kgr;B activity in a patient, comprising administering to a patient a pharmaceutical composition as described above.
Within further aspects, the present invention provides methods for treating a patient afflicted with a disorder associated with NF-&kgr;B activation, comprising administering to a patient a therapeutically effective amount of a pharmaceutical composition as described above, and thereby treating a disorder associated with NF-&kgr;B activation. Such disorders include inflammatory diseases, autoimmune diseases, cancer and viral infection.
Within further asp

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