Attenuation of Murine Collagen-Induced Arthritis by a Novel, Potent, Selective Small Molecule Inhibitor of I B Kinase 2, TPCA-1 (2-[(Aminocarbonyl)amino]-5-(4-fluorophenyl)-3- thiophenecarboxamide), Occurs via Reduction of Proinflammatory Cytokines and Antigen-Induced T Cell Proliferation

Demonstration that I B kinase 2 (IKK-2) plays a pivotal role in the nuclear factorB-regulated production of proinflammatory molecules by stimuli such as tumor necrosis factor (TNF)and interleukin (IL)-1 suggests that inhibition of IKK-2 may be beneficial in the treatment of rheumatoid arthritis. In the present study, we demonstrate that a novel, potent (IC50 17.9 nM), and selective inhibitor of human IKK-2, 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1), inhibits lipopolysaccharide-induced human monocyte production of TNF, IL-6, and IL-8 with an IC50 170 to 320 nM. Prophylactic administration of TPCA-1 at 3, 10, or 20 mg/kg, i.p., b.i.d., resulted in a dosedependent reduction in the severity of murine collagen-induced arthritis (CIA). The significantly reduced disease severity and delay of disease onset resulting from administration of TPCA-1 at 10 mg/kg, i.p., b.i.d. were comparable to the effects of the antirheumatic drug, etanercept, when administered prophylactically at 4 mg/kg, i.p., every other day. Nuclear localization of p65, as well as levels of IL-1 , IL-6, TNF, and interferon, were significantly reduced in the paw tissue of TPCA-1and etanercept-treated mice. In addition, administration of TPCA-1 in vivo resulted in significantly decreased collagen-induced T cell proliferation ex vivo. Therapeutic administration of TPCA-1 at 20 mg/kg, but not at 3 or 10 mg/kg, i.p., b.i.d., significantly reduced the severity of CIA, as did etanercept administration at 12.5 mg/kg, i.p., every other day. These results suggest that reduction of proinflammatory mediators and inhibition of antigen-induced T cell proliferation are mechanisms underlying the attenuation of CIA by the IKK-2 inhibitor, TPCA-1. Rheumatoid arthritis (RA) is a disease characterized by chronic inflammation of the joint, leading to progressive destruction of cartilage and bone. Migration of leukocytes to the synovium results in synovial hypertrophy and the production of proinflammatory mediators by both synoviocytes and leukocytes. These mediators are believed to be responsible for the subsequent cartilage destruction and bone erosion that characterizes the disease (Kingsley and Panayi, 1997; Hasunuma et al., 1998). Many of the proinflammatory molecules associated with RA, including TNF, IL-1, IL-6, IL-8, IFN, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, cyclooxygenase (COX)-2, inducible nitricoxide synthase, matrix metalloproteinase (MMP)-1, and MMP-9, are regulated by the Rel/NFB family of transcription factors (Pahl, 1999). Thus, members of this signaling pathway are potential targets for the development of novel RA therapeutics. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.104.074484. ABBREVIATIONS: RA, rheumatoid arthritis; IKK, I B kinase; NFB, nuclear factorB; TNF, tumor necrosis factor; IL, interleukin; TPCA-1, 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide; LPS, lipopolysaccharide; CIA, collagen-induced arthritis; IFN, interferon; COX, cyclooxygenase; MMP, matrix metalloproteinase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DMSO, dimethyl sulfoxide; DMA, dimethylacetoacetamide; LNC, lymph node cells; ELISA, enzyme-linked immunosorbent assay; JNK3, c-Jun N-terminal kinase 3; AUC, area under the curve; NSAIDs, nonsteroidal anti-inflammatory drugs; DMARDs, disease-modifying antirheumatic drugs; GST, glutathione S-transferase; BSA, bovine serum albumin; DTT, dithiothreitol; HBSS, Hanks’ balanced salt solution; APC, antigen-presenting cells; Th1, subset 1 of T helper cells. 0022-3565/05/3121-373–381$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 312, No. 1 Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics 74484/1181064 JPET 312:373–381, 2005 Printed in U.S.A. 373 at A PE T Jornals on Sptem er 3, 2017 jpet.asjournals.org D ow nladed from In mammals, the Rel/NFB family consists of p50/p105 (NFB1), p52/p100 (NFB2), p65 (RelA), c-Rel (Rel), and RelB, which exist in the cell cytoplasm as homodimeric or heterodimeric complexes. The NFB dimer (classically p50/ p65) is retained in the cytoplasm in an inactive form through its association with I B (inhibitor of NFB) proteins. A variety of stimuli, including TNFand IL-1, are capable of inducing NFB activation. These agents initiate a signaling cascade leading to the phosphorylation of two N-terminal serine residues in I B, which facilitates the ubiquitination and subsequent degradation of I B by the 26S proteosome. Once released from I B, NFB translocates to the nucleus, where it binds to a B consensus sequence encoded within its target gene and initiates transcription (Makarov, 2001; Tak and Firestein, 2001). Because the enzymes responsible for the ubiquitination of phosphorylated I B are constitutively active, the phosphorylation of I B is a critical regulatory step in I B degradation and subsequent NFB activation. This phosphorylation event is catalyzed by the I B kinase (IKK) complex, which consists of two enzymatically active kinases, IKK-1 (IKK ) and IKK-2 (IKK ), and a regulatory subunit, NEMO (IKK ) (Karin, 1999). Divergent physiological roles for the two kinases are suggested by targeted gene deletion studies in which IKK-2-deficient mice, but not IKK-1-deficient mice, exhibited significantly impaired TNFand IL-1-induced NFB activation and IL-6 production (Li et al., 1999; Tanaka et al., 1999). These results suggest that IKK-2, rather than IKK-1, plays a critical role in the NFB-regulated production of proinflammatory molecules induced by stimuli such as TNFand IL-1 and thus is a relevant target for the development of an anti-inflammatory therapeutic. Much evidence indicates a pivotal role for NFB in the etiology of RA. Nuclear localization of p50 and p65 has been shown to be significantly increased in synovial tissue from RA patients, compared with synovium from normal controls (Handel et al., 1995; Han et al., 1998). Similarly, it was demonstrated that fibroblast-like synoviocytes from RA synovium contain constitutively active NFB and spontaneously produce large quantities of IL-6, unlike fibroblast-like synoviocytes from osteoarthritis synovium (Miyazawa et al., 1998). In addition, a number of antirheumatic agents, including glucocorticoids, sulfasalazine, gold salts, leflunomide, and aspirin, are inhibitors of NFB activation (Makarov, 2001; Tak and Firestein, 2001), which may explain, at least in part, their anti-inflammatory effects. Consistent with the data from human synovial tissue, increased NFB binding activity has been demonstrated in the synovium of mice and rats following the development of CIA, adjuvant arthritis, and streptococcal cell wall-induced arthritis (Tak and Firestein, 2001). Additional evidence implicating NFB in animal models of RA comes from the demonstration that in vivo administration of reagents exerting inhibitory effects at various points along the NFB signaling pathway resulted in a reduction of disease (Tak and Firestein, 2001). The results of studies specifically targeting IKK-2 suggest that this enzyme plays a pivotal role in the NFBmediated inflammatory response underlying arthritis. Intraarticular injection of a wild-type IKK-2 gene into the joints of normal rats resulted in paw swelling and synovial inflammation, whereas transfer of a dominant-negative IKK-2 gene decreased the severity of rat adjuvant arthritis (Tak et al., 2001). These studies suggest that inhibition of IKK-2 is a viable approach to the development of a novel therapeutic for RA. In the current study, we characterize a novel, potent, selective small molecule inhibitor of IKK-2, TPCA-1 (2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide). Prophylactic or therapeutic administration of TPCA-1 significantly reduced the severity of murine CIA. This modulation of disease was accompanied by decreased tissue levels of the proinflammatory cytokines, IL-1 , IL-6, TNF, and IFN, as well as reduced T cell proliferation in response to antigen, suggesting that these mechanisms underlie the inhibition of CIA by TPCA-1. Materials and Methods Synthesis of TPCA-1. TPCA-1 was synthesized at GlaxoSmithKline by the Respiratory and Inflammation Center of Excellence for Drug Discovery (King of Prussia, PA). The 2-amino-5-(4-fluorophenyl)-3-thiophenecarboxamide precursor was prepared by the reaction of 4-fluorophenylacetaldehyde, 2-cyanoacetamide, sulfur, and triethylamine in dimethylformamide at 0°C, allowing the reaction to warm to room temperature overnight (Goudie, 1976). Treatment of 2-amino-5-(4-fluorophenyl)-3-thiophenecarboxamide with chlorosulfonylisocyanate in methylene chloride at 0°C followed by aqueous hydrolysis and subsequent recrystallization from ethanol provided TPCA-1. IKK-2 Assay. Recombinant human IKK-2 (residues 1–756) was expressed in baculovirus as an N-terminal GST-tagged fusion protein, and its activity was assessed using a time-resolved fluorescence resonance energy transfer assay. In brief, IKK-2 (5 nM final) diluted in assay buffer (50 mM HEPES, 10 mM MgCl2, 1 mM CHAPS, pH 7.4, with 1 mM DTT and 0.01% w/v BSA) was added to wells containing various concentrations of compound or dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO) vehicle (3% final). The reaction was initiated by the addition of GST-I B substrate (25 nM final)/ATP (1 M final), in a total volume of 30 l. The reaction was incubated for 30 min at room temperature, then terminated by the addition of 15 l of 50 mM EDTA. Detection reagent (15 l) in buffer (100 mM HEPES, pH 7.4, 150 mM NaCl, and 0.1% w/v BSA) containing antiphosphoserine-I B -32/36 monoclonal antibody 12C2 (Cell Signaling Technology Inc., Beverly, MA), labeled with W-1024 europium chelate (PerkinElmer Wallac, Turku, Finland), and an allophycocyanin-labeled anti-GST antibody (ProZyme, San Leandro, CA) was added, and the reaction was further incubated for 60 min at room temperature. The degree of phosphorylation of GST-I B was measured as a ratio of specific 665-nm energy transfer signal to reference europium 620-nm signal, using a Packard Discovery plate reader (PerkinElmer Life Sciences, Buckinghamshire, UK). Lipopolysaccharide-Induced Cytokine/Chemokine Production by Human Monocytes. Human monocytes were isolated from heparinized whole blood by positive selection using CD14 microbeads (Miltenyi Biotec Inc., Auburn, CA). In brief, human whole blood was collected from healthy volunteers and diluted with an equal volume of HBSS (without Ca or Mg ) containing 1 mM EGTA. Diluted blood was layered on a Ficoll-Hypaque gradient (Amersham Biosciences AB, Uppsala, Sweden) and centrifuged at 900g for 30 min. The resulting interface was removed and washed twice with HBSS containing 1 mM EGTA. Cell pellets were resuspended at 1.25 10 cells/ml in PBS containing 0.5% BSA and 2 mM EDTA. Cells were labeled with 20 l CD14 microbeads per 1 10 cells and incubated for 15 min at 6°C with frequent mixing. Labeled cells were washed once and resuspended at 1 10 cells/ml in chilled PBS containing 0.5% BSA and 2 mM EDTA. Cells were applied to magnetized columns using an autoMACS (Miltenyi Biotec Inc.), and separation was performed according to the manufacturer’s instruc374 Podolin et al. at A PE T Jornals on Sptem er 3, 2017 jpet.asjournals.org D ow nladed from tions. The resulting monocyte population was 90% pure as assessed by differential staining. Purified monocytes were washed twice with PBS containing 0.5% BSA and 2 mM EDTA and resuspended to 1 10 cells/ml in warm RPMI 1640 medium containing 10% FBS and L-glutamine. Monocytes were plated at 5 10 cells/well in 48-well tissue culture plates and incubated for 2 h at 37°C. Adhered monocytes were washed once with warm RPMI 1640 medium containing 10% fetal calf serum and L-glutamine, and TPCA-1 in 100% DMSO was added (0.1% DMSO final concentration). The monocytes were incubated with compound for 30 min at 37°C and then stimulated with 200 ng/ml LPS for 24 h. Plates were centrifuged at 500g for 10 min, and supernatants were removed and stored at 20°C until cytokine/chemokine evaluation