NF-κB, a heterodimer of the proteins p50 and RelA, is an inducible eukaryotic transcription factor complex that is broadly expressed and plays a pivotal role in regulating multiple biological responses, such as the inflammatory and immune responses in mammalian cells. See, Baldwin, Jr., Annu. Rev. Immunol. 14:649–683 (1996) and Ghosh, S. et al., Annu. Rev. Immunol. 16:225–260 (1998). NF-κB binds to regulatory regions of genes, termed enhancers, that increase the expression of these genes thereby exerting important effects on inflammatory, immune and anti-apoptotic responses. Activation of NF-κB is implicated in a variety of chronic inflammatory diseases. In addition, the long terminal repeat (LTR) of HIV is subject to activation by NF-κB, a response that can lead to heightened levels of viral replication.
NF-κB was first identified as a constitutively expressed protein that bound to a specific decameric DNA sequence (5′-ggg act ttc c-3′SEQ ID NO:1), within the intronic enhancer of the immunoglobulin kappa light chain in mature B- and plasma cells, but not pre B-cells (Sen et al. (1986) Cell 46:705–716). Later, it was demonstrated that the DNA binding activity of NF-κB is induced in most cells following exposure to a variety of exogenously applied stimuli, and that this activation occurs independently of de novo protein synthesis. NF-κB binds to specific sites on DNA (referred to as κB sites, and having the general consensus sequence: 5′-ggg rnn tyc c-3′ (SEQ ID NO:2), where R is a purine, and Y is a pyrimidine). These κB sites have been identified in promoters and enhancers of a large number of inducible NF-κB genes. For reviews see, e.g., May et al., Sem. Cancer Biol. 8:63–73 (1997); and May et al., Immunol. Today 19(2):80–88 (1998).
The prototypical NF-κB complex, which corresponds to a heterodimer of p50 and RelA subunits, is kept in an inactive form and sequestered in the cytoplasm by a family of inhibitory proteins termed the IκBs, which includes IκBα. Upon exposure to a wide variety of stimuli, for example proinflammatory cytokines like tumor necrosis factor α (TNF-α), and interleukin 1 (IL-1), IκBα is phosphorylated. This event, mediated by a macromolecular IκB kinase complex (IKK) (Karin, M., Oncogene 18:6867–6874 (1999)), triggers the rapid ubiquitination and subsequent degradation of this inhibitor by the 26S proteasome complex. The unmasking of the nuclear localization signal in the RelA component of the p50/RelA NF-κB heterodimer allows its rapid translocation into the nucleus, where it engages cognate κB sites and activates transcription of various target genes.
Transcriptional activation by NF-κB principally reflects the action of its RelA subunit, which contains an active C-terminal transcriptional activation domain. In contrast, the p50 subunit principally plays a role in DNA binding. Overall transcriptional activity of the RelA subunit is also regulated by casein kinase II- and IKK-mediated phosphorylation of serine residues present in the C-terminal activation domain of RelA. See, for example, Sakurai, H., J. Biol. Chem. 274:30353–30356 (1999), Wang, D., J. Biol. Chem. 275:32592–32597 (2000), and Bird, T. A., J. Biol. Chem. 272:32606–32612 (1997).
Binding of NF-κB to DNA promotes the recruitment of such co-activators as p300 or CBP (cyclic AMP Response Element Binding Protein (CREB) Binding Protein) and P/CAF that participate in the transcriptional activation of target genes. See, Perkins, N. D. et al., Science 275:523–527 (1997), Hottiger, M. O., EMBO J. 17:3124–3134 (1998), and Sheppard, K. A. et al., Mol. Cell. Biol. 19:6367–6378 (1999). Phosphorylation of the N-terminal portion of the RelA subunit by protein kinase A facilitates its assembly with CBP/p300 (Zhong, H. et al., Mol. Cell 1:661–671 (1998)). Both CBP and p300 exhibit histone acetyltransferase activity, which has been implicated in the control of gene expression. This action involves both acetylation of core histones that lead to changes in chromatin structure, and direct acetylation of select host transcription factors like p53, GATA-1 and E2F, where acetylation alters the biological function of the factors. See, for example, Imhof, I. and Wolffe, A. P., Curr. Biol. 8:422–424 (1998); Sterner, D. E. and Berger, S. L., Microbiol. Mol. Biol. Rev. 64:435–459 (2000); and Kouzarides, M. T., EMBO J. 19:1176–1179 (2000).
Although the biochemical steps underlying IκB degradation and NF-κB activation are relatively well understood, much less is known about how the cell re-establishes control over the powerfully active nuclear NF-κB complexes once they are expressed in the nucleus.
Histones are proteins found in the nucleus that play a major role in regulating gene expression by modifying chromatin structure. See, Imhof, A. and Wolffe, A. P., Curr. Biol. 8:422–424 (1998). Histones are acetylated and deacetylated in vivo. These opposing processes are regulated by two complementary groups of enzymes, the histone acetyltransferases (HATs) and histone deacetylases (HDACs). Eight different HDACs (HDAC 1–8) have now been identified in mammalian cells. See, Cress, W. D. and Seto, E., J. Cell Physiol. 184:1–16 (2000). Based on their primary structure, these HDACs have been divided into two classes; the class I HDACs include HDAC 1, 2, 3 and 8, which share homology with a yeast transcriptional repressor, RPD3 (Taunton, J. et al., Science 272:408–411 (1996)), and the class II HDACs including HDAC 4, 5, 6 and 7, which display similarity to another yeast deacetylase, HDA1 (Rundlett, S. E. et al., Proc. Natl. Acad. Sci. USA 93:14503–14508 (1996)). Each of the HDACs contains a conserved catalytic domain that mediates deacetylation of histones (Ng, H. H. and Bird, A., TIBS 25:121–126 (2000)). In general, acetylation of histones by the HATs promotes nucleosome rearrangement and transcriptional activation. Conversely, deacetylation of histones by the HDACs promotes nucleosome assembly and transcriptional repression. See, Kouzarides, M. T., EMBO. J. 19:1176–1179 (2000) and Kuo, H. and Allis, C. D., Bioessays, 20:615–626 (1998). In addition, it is known that trichostatin A (TSA) is a specific inhibitor of the HDACs. See, Yoshida, M. and Horinouchi, S., Ann N.Y. Acad. Sci. 886:23–36 (1999).
Since NF-κB plays a key role in regulating both inflammatory and immune responses in mammalian cells, as well as contributing to cancer cell growth and increased replication of various viruses like HIV-1, the development of agents that impair NF-κB activation or function could have important therapeutic applications. The present invention describes a novel approach to the regulation of nuclear NF-κB activity taking advantage of the recent observation that nuclear NF-κB action is regulated by reversible acetylation.
There is a need for compositions and methods to modulate NF-κB activity, particularly agents that modulate NF-κB activity through a mechanism or pathway different from the NE-κB modulating agents currently available. Providing such agents expands the scope of methods for treating conditions associated with dysregulation of NF-κB activity, thus ultimately providing the clinician and the patient with alternative therapies. Thus, there is a need for methods for identifying agents that can modulate NF-κB activity through, for example, a pathway other than that involving regulation of interaction with IκBα and its components, or other extranuclear pathway. The present invention addresses this need.
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