**3.1 Activation of NF-κB in RA**

NF-κB is one of the best-characterized transcription factors and regulates the expression of many genes, most of which encode proteins that play crucial roles in the processes of immunity and inflammation. The activation of NF-ĸB has been associated with the upregulation of pro-inflammatory genes involved in several inflammatory conditions [Baldwin, 1996], and has been implicated in pathogenesis of RA [Firestein, 2004]. NF-κB activation has been studied in animal models of arthritis [Han et al., 1998; Palombella et al., 1998] and in the synovium of RA patients [Handel et al., 1995; Firestein, 2004]. NF-κB is essential for TNF-induced synovial cell activation and proliferation as several studies indicated that treatment of synovial cells with an antioxidant agent inhibited TNF-α induced NF-κB activation and transcription [Fujisawa et al., 1996]. Moreover, nuclear extracts from IL-1β stimulated human synovial broblasts contained p65 DNA-binding

Fig. 5. Activation of Nuclear factor-ĸB by the proteasome system

drug research in recent years [Elliott et al., 2003].

**3.1 Activation of NF-κB in RA** 

NF-κB promotes transcription of genes which encode cytokines (TNF-α, IL-6, IL-1), stress response factors (Cyclooxygenase-2, NO), cell cycle regulators, and anti-apoptotic proteins (IAP-1, Bcl-2 family) [Delhalle et al., 2004]. The pathological activation of NF-κB is a cause of many inflammatory diseases including RA and has been an important target for therapeutic

NF-κB is one of the best-characterized transcription factors and regulates the expression of many genes, most of which encode proteins that play crucial roles in the processes of immunity and inflammation. The activation of NF-ĸB has been associated with the upregulation of pro-inflammatory genes involved in several inflammatory conditions [Baldwin, 1996], and has been implicated in pathogenesis of RA [Firestein, 2004]. NF-κB activation has been studied in animal models of arthritis [Han et al., 1998; Palombella et al., 1998] and in the synovium of RA patients [Handel et al., 1995; Firestein, 2004]. NF-κB is essential for TNF-induced synovial cell activation and proliferation as several studies indicated that treatment of synovial cells with an antioxidant agent inhibited TNF-α induced NF-κB activation and transcription [Fujisawa et al., 1996]. Moreover, nuclear extracts from IL-1β stimulated human synovial broblasts contained p65 DNA-binding NF-κB complexes and both the NF-κB classical oligonucleotide decoy and antisense oligonucleotide specic to p65, and they produced a concentration dependent decrease in IL-1-stimulated PGE2 production [Handel, 1995]. Additionally, NF-κB activator, IL-18 can indirectly stimulate osteoclast formation through up-regulation of RANKL production from T cells in RA synovitis [Dai et al., 2004]. Blocking of IKKβ *in vitro* with a dominant negative adenoviral construct was shown to inhibit the induction of IL-6, IL-8, and intercellular adhesion molecule-1 (ICAM-1) after stimulation with IL-1 or TNF-α [Aupperle et al., 2001].

The signicance of NF-κB in inammatory joint disease has been validated by numbers of arthritis models such as carrageenan-induced paw edema, collagen-induced arthritis and adjuvant-induced arthritis [Min et al, 2009, Campo et al, 2011, Ahmed et al., 2010]. In animal models of arthritis the activation of NF-κB appears to precede the onset of disease, and the blockade of NF-κB decreases arthritis severity [Tsao et al., 1997; Ahmed et al., 2010]. Intraarticular gene transfer of IKKβ-wild type into the joints of normal rats resulted in signicant paw swelling and accompanied synovial inammation. Increased IKK activity was detectable in the IKKβ-wt-injected ankle joints which was coincident with enhanced NF-κB-DNA-binding activity. Intra-articular gene transfer of IKKβ-dominant negative signicantly ameliorated the severity of adjuvant arthritis, accompanied by a signicant decrease in NFκB DNA expression in the joints of adenoviral IKKb-dominant negative-treated animals [Tak et al., 2001].

### **3.1.1 NF-κB in RA joint destruction**

Progressive destruction of bone and articular cartilage plays a pivotal role in the pathogenesis of RA. During joint inflammation, the inflamed synovium forms a pannus tissue, which grows into the bone and causes destruction, initially as marginal erosions at the site of synovial proliferation where bone is unprotected by hyaline cartilage. Subsequent bone destruction leads to sublaxation and deformity. Cytokines such as TNF-α, IL-1β and IL-17 stimulate the activation of bone destroying osteoaclasts, and the production of destructive proteases - matrix metalloproteinases (MMPs). MMPs have been suggested to be involved in the pathogenesis of RA and OA through their ability to degrade proteoglycans [Flannery et al., 1992; Humbry et al., 1995].

NF-κB is essential for osteoclast formation and survival through the receptor activator of the nuclear factor kappa-B ligand (RANKL) pathway [Soysa et al., 2009]. Abnormal activation of NF-κB signalling in osteoclasts has been observed in osteolytic conditions, including arthritis, Paget's disease of bone, and periodontitis [Xu et al., 2009]. Inhibition or deletion of RANKL prevents bone destruction [Zwerina et al., 2004]. Further, it is demonstrated that inhibition of IκB-kinase complex can suppress RANKL stimulated NF-κB activation and osteoclastogenesis both *in vitro* and *in vivo*. Additionally, this peptide signicantly reduced the severity of collagen-induced arthritis in mice by reducing levels of TNF-α and IL-1β, and thereby abrogating joint swelling and reducing destruction of bone and cartilage [Jim et al., 2004]. Elevated levels of MMP-1 (collagenase-1) in the synovial fluid and serum of RA patients [Green et al., 2003; Yamanaka et al., 2000] and MMP-3 (stromelysin) in the synovial fluid from RA patients has been determined [Hembry et al., 1995]. Interestingly, it has been reported that NF-κB regulates synthesis of MMPs including MMP-I and MMP-3 [Thurberg et al., 1998].

Proteasome Targeted Therapies in Rheumatoid Arthritis 141

Peptide aldehydes were the first proteasome inhibitors to be developed [Palombella et al., 1994; Rock et al., 1994]. These include MG132 (Z-Leu-Leu- Leucinal-) (Fig 5), MG115 (Z-Leu-Leu-norvalinal-) and calpain inhibitor I (*N*-acetyl-Leu-Leu-norleucinal). These compounds are potent, reversible and cell permeable. MG132 is a reversible inhibitor of the

Boronate inhibitors are much more potent than their structurally analogous peptide aldehydes [Adams et al., 1998]. These includes MG262 (Z-Leu-Leu-Leu-boronate; analogous to MG132) and PS-341 (pyrazylcarbonyl-Phe-Leu-boronate; analogous to the aldehyde PS-402). MG262 is a cell permeable and reversible inhibitors of the chymotrypsin like activity of the proteasome. PS-341 is clinically the most advanced proteasome inhibitor and inhibits the chymotrypsin like active site of the proteasome β-subunit. Its boronic acid group binds the

Lactacystin is a naturally occurring compound produced by *Streptomyces lactacystinaeus.* It selectively targets the β5 subunit of the proteasome [Fenteany et al., 1995] by covalent acylation of the amino-terminal threonine residues and is considered as an irreversible inhibitor of the proteasome. The active component of lactacystin is the highly reactive *clasto-*

active site threonine in the proteasome with high affinity and specificity (Fig 5).

Fig. 6. Structures of selected proteasome inhibitors [Elliott et al., 2003].

**5.1.1 Peptide aldehydes** 

**5.1.2 Boronic acid peptides** 

**5.1.3 Lactacystin** 

chymotrypsin like activity of the proteasome.

lactacystin β-lactone and PS-519 (Fig 5).
