Abstract
IκB kinase (IKK) β is essential for inflammatory cytokine-induced activation of nuclear factor κB (NF-κB). NF-κB plays a pivotal role in the function of major cell types that contribute to the pathophysiological process of rheumatoid arthritis (RA). Here, we report the mechanism and the effect of the IKKβ inhibitor N-(6-chloro-7-methoxy-9H-β-carbolin-8-yl)-2-methylnicotinamide (ML120B), a β-carboline derivative, on NF-κB signaling and gene activation in RA-relevant cell systems. ML120B is a potent, selective, reversible, and ATP-competitive inhibitor of IKKβ with an IC50 of 60 nM when evaluated in an IκBα kinase complex assay. ML120B does not inhibit other IKK isoforms or a panel of other kinases. ML120B concentration-dependently inhibits tumor necrosis factor α (TNFα)-stimulated NF-κB signaling via inhibition of IκBα phosphorylation, degradation, and NF-κB translocation into the nucleus. For the first time, we have demonstrated that in human fibroblast-like synoviocytes, TNFα- or interleukin (IL)-1β-induced monocyte chemoattractant protein-1 regulated on activation, normal T cell expressed and secreted and production is IKKβ-dependent. In addition, for the first time, we have demonstrated that lipopolysaccharide- or peptidoglycan-induced cytokine production in human cord blood-derived mast cells is IKKβ-dependent. In addition, in human chondrocytes, ML120B inhibited IL-1β-induced matrix metalloproteinase production with an IC50 of approximately 1 μM. ML120B also blocked IL-1β-induced prostaglandin E2 production. In summary, ML120B blocked numerous NF-κB-regulated cell responses that are involved in inflammation and destructive processes in the RA joint. Our findings support the evaluation of IKKβ inhibitors as anti-inflammatory agents for the treatment of RA.
Footnotes
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This work was presented as a poster in Keystone Symposia NF-κB: Biology and Pathology. UT, 2004 [Wen D, Gangurde P, Morgan JG, Nong Y, Savinainen A, Kujawa J, Schopf L, Hepperle M, Harriman G, Jaffee B, et al. (2004) NF-κB regulation by IKKβ in rheumatoid arthritis Poster 365.].
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ML120B was covered in the published Parent Cooperation Treaty patent application WO 2004092167: Hepperle ME, Liu JF, Soucy F, Ye Y, Murray RS, Prakash R, Little JD, Castro A, Mazdiyasni H, Fleming PE, et al. (2004) inventors, Millennium Pharmaceuticals, Inc., assignee. Preparation of substituted β-carboline IκB kinase 2 (IKK-2) inhibitors as potential anti-inflammatory, immunomodulatory, or anticancer agents.
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doi:10.1124/jpet.105.097584.
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ABBREVIATIONS: NF-κB, nuclear factor κB; RA, rheumatoid arthritis; TNF, tumor necrosis factor; IL, interleukin; MCP-1, monocyte chemoattractant protein-1; RANTES, regulated on activation, normal T cell expressed and secreted; MMP, matrix metalloproteinase; IKK, IκB kinase; ML120B, N-(6-chloro-7-methoxy-9H-β-carbolin-8-yl)-2-methylnicotinamide; LPS, lipopolysaccharide; PBMC, peripheral blood mononuclear cell; HFLS, human fibroblast-like synoviocyte(s); MC, mast cell; PGE, prostaglandin E; BSA, bovine serum albumin; PS1145, N-(6-chloro-9H-β-carbolin-8-yl) nicotinamide; DMSO, dimethyl sulfoxide; PMA, phorbol myristate acetate; PGN, peptidoglycan; GST, glutathione S-transferase; AP-1, activator protein-1; MEKK, mitogen-activated protein kinase kinase kinase; PKC, protein kinase C; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence-activated cell sorting; PE, phosphatidylethanolamine; TLR, toll-like receptor; wt, wild type; DN, dominant-negative; SR, super-repressor; GFP, green fluorescent protein; ELAM, endothelial cell leukocyte adhesion molecule (E-selectin); LDH, lactate dehydrogenase; MAP, mitogen-activated protein; COX, cyclooxygenase; m.o.i., multiplicity of infection.
- Received October 25, 2005.
- Accepted March 6, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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