Abstract
Nuclear factor (NF)-κB activation has been clearly linked to the pathogenesis of multiple inflammatory diseases including arthritis. The central role that IκB kinase-2 (IKK-2) plays in regulating NF-κB signaling in response to inflammatory stimuli has made this enzyme an attractive target for therapeutic intervention. Although diverse chemical classes of IKK-2 inhibitors have been identified, the binding kinetics of these inhibitors has limited the scope of their applications. In addition, safety assessments of IKK-2 inhibitors based on a comprehensive understanding of the pharmacokinetic/pharmacodynamic relationships have yet to be reported. Here, we describe a novel, potent, and highly selective IKK-2 inhibitor, PHA-408 [8-(5-chloro-2-(4-methylpiperazin-1-yl)isonicotinamido)-1-(4-fluorophenyl)-4,5-dihydro-1H-benzo[g]indazole-3-carboxamide]. PHA-408 is an ATP-competitive inhibitor, which binds IKK-2 tightly with a relatively slow off rate. In arthritis-relevant cells and animal models, PHA-408 suppresses inflammation-induced cellular events, including IκBα phosphorylation and degradation, p65 phosphorylation and DNA binding activity, the expression of inflammatory mediators, and joint pathology. PHA-408 was efficacious in a chronic model of arthritis with no adverse effects at maximally efficacious doses. Stemming from its ability to bind tightly to IKK-2, as a novelty, we demonstrated that PHA-408-mediated inhibition of IKK-2 activity correlated very well with its ability to modulate the fate of IKK-2 substrates and downstream transcriptional events. We ultimately directly linked IKK-2 activity ex vivo and in vivo to markers of inflammation with the inhibitor plasma concentrations. Thus, PHA-408 represents a powerful tool to further gain insight into the mechanisms by which IKK-2 regulates NF-κB signaling and validates IKK-2 as a therapeutic target.
Footnotes
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G.M. and C.D.S. contributed equally to this work.
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doi:10.1124/jpet.108.143800.
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ABBREVIATIONS: NF, nuclear factor; IKK, IκB kinase; NEMO, NF-κB essential modulator; PHA-408, 8-(5-chloro-2-(4-methylpiperazin-1-yl)isonicotinamido)-1-(4-fluorophenyl)-4,5-dihydro-1H-benzo[g]indazole-3-carboxamide; BSA, bovine serum albumin; LPS, lipopolysaccharide; HSP, heat shock protein; ELISA, enzyme-linked immunosorbent assay; TNF, tumor necrosis factor; PG, prostaglandin; MAPK, mitogen-activated protein kinase; JNK, c-Jun NH2-terminal kinase; DMEM, Dulbecco's modified Eagle's medium; DPBS, Dulbecco's phosphate-buffered saline; IL, interleukin; 5-FAM, 5-carboxyfluorescein; SEAP, secreted alkaline phosphatase; rh, recombinant human; Me2SO, dimethyl sulfoxide; RASF, rheumatoid arthritis-derived synovial fibroblast; PBMC, peripheral blood mononuclear cell; SCW, streptococcal cell wall; EMSA, electrophoretic mobility shift assay; HWB, human whole blood; CT, computed tomography; AUC, area under the curve; SC-806, 1-[5-(4-chlorophenyl)-4-(4-pyridinyl)-1H-pyrazol-3-yl]-4-methylpiperazine; BAY 11-7082, (E)-3-[(4-methylphenylsulfonyl]-2-propenenitrile.
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↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
- Received July 23, 2008.
- Accepted January 22, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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