A selective novel low‐molecular‐weight inhibitor of IκB kinase‐β (IKK‐β) prevents pulmonary inflammation and shows broad anti‐inflammatory activity

Pulmonary inflammatory diseases such as asthma are characterized by chronic, cell-mediated inflammation of the bronchial mucosa. Recruitment and activation of inflammatory cells is orchestrated by a variety of mediators such as cytokines, chemokines, or adhesion molecules, the expression of which is regulated via the transcription factor nuclear factor kappa B (NF-κB). NF-κB signaling is controlled by the inhibitor of kappa B kinase complex (IKK), a critical catalytic subunit of which is IKK-β. We identified COMPOUND A as a small-molecule, ATP-competitive inhibitor selectively targeting IKK-β kinase activity with a Ki value of 2 nM. COMPOUND A inhibited stress-induced NF-κB transactivation, chemokine-, cytokine-, and adhesion molecule expression, and T- and B-cell proliferation. COMPOUND A is orally bioavailable and inhibited the release of LPS-induced TNF-α in rodents. In mice COMPOUND A inhibited cockroach allergen-induced airway inflammation and hyperreactivity and efficiently abrogated leukocyte trafficking induced by carrageenan in mice or by ovalbumin in a rat model of airway inflammation. COMPOUND A was well tolerated by rodents over 3 weeks without affecting weight gain. Furthermore, in mice COMPOUND A suppressed edema formation in response to arachidonic acid, phorbol ester, or edema induced by delayed-type hypersensitivity. These data suggest that IKK-β inhibitors offer an effective therapeutic approach for inhibiting chronic pulmonary inflammation. Keywords: Protein kinases, asthma, inflammation, lung, signal transduction, transcription factors Introduction Asthma is a chronic inflammatory disease with increasing incidence worldwide (Beasley, 2002). While mild to moderate asthma is well controlled by inhaled glucocorticoids (GCs) and β2-agonists, about 5% of patients do not well respond to this treatment. Despite this small percentage, these patients contribute disproportionally high (about 50%) to the overall health-care cost of asthma (Adcock & Ito, 2004). Little is known about the underlying pathology of steroid-resistant asthma, but it seems that one-half to two-thirds of severe asthmatics show persistent large airway tissue eosinophilia and other signs of inflammation despite continued high-dose steroid treatment (Wenzel, 2003). On a molecular level, it is still unclear how exactly GCs exert their anti-inflammatory activity and, consequently, steroid resistance is not well understood. Recent evidence indicates that a major part of the anti-inflammatory effects of GCs is based on transrepressing and therefore inhibiting the proinflammatory transcription factors activator protein-1 (AP-1) or nuclear factor kappa B (NF-κB). One hypothesis for steroid insensitivity is increased activation/expression of AP-1 or NF-κB proteins which cannot be sufficiently transrepressed by GC-bound glucocorticoid receptor (GR) (De Bosscher et al., 2003; Leung & Bloom, 2003). Alternative hypotheses include the reduced ability of the GR to bind to the ligand or increased expression of an alternatively spliced, dominant-negative GR (GR-β). The first hypothesis is supported by a recent study showing that persistent activation of NF-κB signaling is observed in severe, uncontrolled asthma (Gagliardo et al., 2003). In addition, a variety of data both from asthma patients as well as from deletion mutants of NF-κB proteins in mice support a critical role for NF-κB in asthma pathology (Yang et al., 1998; Donovan et al., 1999; Christman et al., 2000). Inhibition of NF-κB activity might therefore be an effective alternative approach to treat asthma, including severe forms refractory to GCs. NF-κB proteins are a family of ubiquitously expressed transcription factors that, in mammals, consist of five members: p65 (RelA), RelB, c-Rel, NF-κB1 (p50 and its precursor 105) and NF-κB2 (p52 and its precursor p100) (Verma et al., 1995). NF-κB and related family members are involved in the regulation of more than 50 genes, which are activated upon inflammatory and immune responses (Baeuerle & Baichwal, 1997). NF-κB shows a unique mode of regulation: It is kept in an inactive state in the cytoplasm by interacting with members of the IκB family of proteins which mask the nuclear translocation signal of NF-κB. Upon stimulation of cells by various cytokines (e.g. TNF-α, IL-1β), CD40 ligand, lipopolysaccharide (LPS), oxidants, mitogens (e.g. phorbol ester) or viruses, IκB proteins become phosphorylated at specific serine residues by the inhibitor of κB (IKK) kinase complex. This triggers poly-ubiquitinylation and subsequent degradation through a proteasome-dependent pathway, resulting in transcriptionally active NF-κB (Verma et al., 1995; Baeuerle & Baichwal, 1997; Rothwarf & Karin, 1999; Gosh & Karin, 2002; Yamamoto & Gaynor, 2004). IKK activity resides in a high-molecular-weight complex comprising of at least two catalytic subunits, IKK-α (IKK1) and IKK-β (IKK2), and the associated regulatory subunit IKK-γ/NEMO (Rothwarf & Karin, 1999; Gosh & Karin, 2002; Yamamoto & Gaynor, 2004). Although IKK-α and IKK-β have a high degree of sequence homology and share similar structural domains, IKK-β has a 20–50-fold higher level of kinase activity for IκB than does IKK-α (Li et al., 1998). More dramatic differences became obvious by the generation of IKK-α- and IKK-β-deficient mice. ikk-α−/− mice presented an unexpected phenotype including shorter limbs and skull, and a fused tail, all enveloped in a shiny and sticky skin (Hu et al., 1999; 2001; Takeda et al., 1999; Sil et al., 2004). These mice die perinatally and have hyperproliferative epidermal cells that do not differentiate. However, IL-1β- and TNF-α-induced NF-κB activation is normal, and the phosphorylation and degradation of IκB proteins is also unchanged. Thus, IKK-α is involved in dermal and skeletal development and cannot be compensated for by IKK-β. Furthermore, it was recently shown that IKK-α plays a role in B-cell maturation and secondary lymphoid organ formation through processing of the NF-κB2 precursor p100 (Senftleben et al., 2001; Muller & Siebenlist, 2003). On the other hand, IKK-β-deficient mice (ikk-β−/−) die as embryos and show massive liver degeneration due to hepatocyte apoptosis (Li QT et al., 1999; Li ZW et al., 1999; Tanaka et al., 1999). In these mice, marked defects in the activation of the NF-κB pathway triggered by pro-inflammatory cytokines such as TNF-α became obvious (Tanaka et al., 1999). Although IKK-α and -γ have also been shown to be involved in NF-κB stimulation by cytokines (Rudolph et al., 2000; Li et al., 2002), it is generally believed that during inflammation IKK-β is more critical than IKK-α in activating the NF-κB pathway and almost all proinflammatory functions reported for NF-κB require the IKK-β subunit for activation. Thus, we aimed at identifying a small-molecule inhibitor selectively targeting IKK-β. This study characterizes the biochemical, cellular and in vivo anti-inflammatory profile of COMPOUND A, a potent and selective inhibitor of IKK-β. Our results highlight the therapeutic potential of such compound for the treatment of asthma and other inflammatory diseases.