A Novel, Highly Selective, Tight Binding IκB Kinase-2 (IKK-2) Inhibitor: A Tool to Correlate IKK-2 Activity to the Fate and Functions of the Components of the Nuclear Factor-κB Pathway in Arthritis-Relevant Cells and Animal Models

Abstract

Nuclear factor (NF)-B activation has been clearly linked to the pathogenesis of multiple inflammatory diseases including arthritis. The central role that IB kinase-2 (IKK-2) plays in regulating NF-B 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, andPHA-408 is an ATPcompetitive 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 IB␣ 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-B signaling and validates IKK-2 as a therapeutic target.The NF-B family of inducible transcription factors regulates the expression of numerous genes, which are central to developmental and immune processes, cell survival, proliferation, and differentiation (Baeuerle and Henkel, 1994). However, dysregulated NF-B activity leads to the onset of several human pathologies, including cancer and inflammatory diseases such as rheumatoid arthritis, asthma, and in-G.M. and C.D.S. contributed equally to this work. Article, publication date, and citation information can be found at

Chemical biology of inflammatory cytokine signaling

“…41,42 The phase I clinical trial of topical formulation of IMD-0354 for treatment of atopic dermatitis has been successfully completed. Other synthetic IKK inhibitors, including S1627, 43 2-benzamido-pyrimidines, 44 2-amino-3,5-diarylbenzamides, 45,46 6-aryl-7-alkoxyisoquinolines, 47 4-phenyl-7-azaindoles, 48 PHA-408 49,50 and PF-184, 50 have been reported.…”

Novel Tight-Binding Inhibitory Factor-κB Kinase (IKK-2) Inhibitors Demonstrate Target-Specific Anti-Inflammatory Activities in Cellular Assays and following Oral and Local Delivery in an in Vivo Model of Airway Inflammation

“…The dissociation rate constants of PHA-408 and PF-184 from rhIKK-2 were determined by monitoring the recovery of IKK-2 kinase activity over time as described in Mbalaviele et al (2009). In brief, the kinase activity of rhIKK-2 was determined by monitoring the phosphorylation of 5FAM-GRHDSGLDSMK (American Peptide Co., Inc., Sunnyvale, CA) with use of the LabChip 3000 (Caliper Life Sciences, Hopkinton, MA).…”

Periodic, Partial Inhibition of IκB Kinase β-Mediated Signaling Yields Therapeutic Benefit in Preclinical Models of Rheumatoid Arthritis

“…We have previously reported that the selective IKK␤ inhibitor BMS-345541 was highly efficacious against both inflammation and joint destruction in collagen-induced arthritis in mice and in a model of inflammatory bowel disease (MacMaster et al, 2003;McIntyre et al, 2003). Subsequently, other IKK inhibitors have also been reported to provide benefit in experimental arthritis models (Podolin et al, 2005;Schopf et al, 2006;Mbalaviele et al, 2009). BMS-345541 has also been shown to be effective in various tumor models and suggests that IKK␤ inhibitors may be beneficial in the treatment of certain cancers in humans (Yang et al, 2006;Ammann et al, 2009).…”