Selective inhibitors of nuclear factor kappab activation and uses thereof

This patent application claims benefit of priority of provisional patent application U.S. Ser. No. 60/517,852, filed Nov. 6, 2003, now abandoned.

This invention was produced in part using funds obtained through a Department of Defense US Army Breast Cancer Research Program grant (BC010610), a PO1 grant (CA91844) from the National Institutes of Health and a P50 Head and Neck SPORE grant from the National Institutes of Health. Consequently, the federal government has certain rights in this invention.

1. Field of the Invention

The present invention relates generally to the molecular biology of nuclear factor-kappa B (NF-κB). More specifically, the present invention relates to polypeptides that can selectively inhibit NF-κB activation, downregulate NF-κB mediated gene expression and enhance apoptosis induced by TNF and other apoptotic stimuli.

2. Description of the Related Art

Nuclear Factor-KB (NF-κB) represents a group of five proteins, namely c-Rel, Rel A (p65), Rel B, NF-kB1 (p50 and p105), and NF-κB2 (p52). NF-κB is regulated by a family of inhibitors called IκB. In an inactive state, NF-κB is present in the cytoplasm as a heterotrimer consisting of p50, p65, and IκBα subunits. In response to an activation signal, the IκBα subunit is phosphorylated at serine residues 32 and 36, ubiquitinated at lysine residues 21 and 22, and degraded through the proteosomal pathway, thus exposing the nuclear localization signals on the p50-p65 heterodimer. The p65 is then phosphorylated, leading to nuclear translocation and binding to specific DNA sequence, which in turns results in transcription of various genes including cyclin D1, cyclooxyenase (COX) 2 and matrix metalloproteinase (MMP) 9.

The p65 subunit of NF-κB, which contains at least two strong transactivation domains (TAD) within the C terminus (TA1 30 amino acid; TA2 90 amino acid), has been shown to undergo phosphorylation upon activation. The sites of phosphorylation and the kinase responsible for p65 phosphorylation remain controversial. For instance, phosphorylation at Ser 276 by protein kinase A, at Ser 529 by casein kinase II, at Ser 536 by IKK-β, and at serine 471 by PKC-ε have been demonstrated. In addition, phosphorylation of p65-TAD by glycogen synthase kinase-3β and by Ca²⁺/calmodulin-dependent protein kinase IV have been demonstrated.

NF-κB has been shown to regulate the expression of a number of genes whose products are involved in inflammation, viral replication, carcinogenesis, anti-apoptosis, invasion and metastasis. These include anti-apoptosis genes, adhesion molecules, chemokines, inflammatory cytokines, and cell cycle regulatory genes. Thus agents that can suppress NF-κB activation have the potential to treat a variety of diseases that involves inflammation, apoptosis and carcinogenesis.

Most proteins enter the cell through their specific cell surface receptors. Recent studies, however, indicate that certain short protein sequences can enter the cells without any receptors and such proteins have been described as protein transduction domain (PTD) peptides (Lindgren et al., 2000; Schwarze and Dowdy, 2000). Most of the protein transduction domain peptides are arginine-rich peptides (Futaki et al., 2003). Importantly, conjugation of proteins, peptides and antisense oligonucleotides to these protein transduction domain peptides has been shown to deliver these cargos effectively, allowing observation of biological action in several cell and animal models (Lindgren et al., 2000; Schwarze and Dowdy, 2000). Peptides derived from third helix of the antennapedia homeodomain, herpes virus structural protein, and HIV tat protein have been used to deliver both small and large peptides of interest to the cells through an energy- and receptor-independent mechanism (Derossi et al., 1994; Elliott and O\'Hare, 1997; Fawell et al., 1994).

Using these protein transduction domain peptides, several peptides based on protein-protein interaction domains have been delivered to the cells to suppress cell signaling. These include Grb2 binding peptide, mitogen-activated protein kinase, STAT3, NEMO-IKK interacting peptide, and peptides carrying nuclear localization sequences. Besides peptides, protein transduction domain peptides have also been used to deliver larger full length polypeptides, including IκBα, cyclin-dependent kinase inhibitory protein p27, anti-apoptotic proteins Bcl-xl, and proapoptotic proteins.

The prior art is deficient in providing a cell permeable inhibitor specific for NF-κB. The present invention fulfills this long-standing need and desire in the art by disclosing the construction of a cell permeable NF-κB-specific inhibitor comprising a NF-κB polypeptide linked to an antennapedia-derived protein transduction domain. This inhibitor can suppress NF-κB activation, suppress NF-κB-mediated gene transcription and enhance apoptosis induced by TNF and other apoptotic stimuli.

The present invention is directed to a cell permeable NF-KB inhibitor comprising (i) a polypeptide of SEQ ID NO. 5 or 11, or homologues or derivatives thereof, and (ii) a protein transduction domain which is able to transport the polypeptide across cell membrane. In general, the protein transduction domain is derived from the third helix of the antennapedia homeodomain, herpes virus structural protein, or HIV tat protein. In one embodiment of the present invention, the protein transduction domain derived from the third helix of the antennapedia homeodomain has the sequence of SEQ ID NO. 3, and the cell permeable. NF-κB inhibitor has the sequences of SEQ ID NO. 4 or 10.

In another aspect, the present invention provides methods of using the NF-κB inhibitor to inhibit DNA binding activity of NF-κB or enhance apoptosis in a cell. In general, DNA binding activity of NF-κB induced by TNF, LPS, IL-1, okadaic acid, PMA, H₂O₂, cigarette smoke condensate, TNF receptor 1 (TNFR1), TNF receptor-associated death domain (TRADD), TNF receptor-associated factor 2 (TRAF2), NF-κB-inducing kinase (NIK), or IκBα kinase (IKK) could be inhibited by the inhibitor, whereas apoptosis induced by TNF, or chemotherapeutic agent such as doxorubicin or cisplatin could be enhanced by the inhibitor.

Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.