Catherine S. Tripp

A Selective IKK-2 Inhibitor Blocks NF-κB-dependent Gene Expression in Interleukin-1β-stimulated Synovial Fibroblasts

NF-κB-induced gene expression contributes significantly to the pathogenesis of inflammatory diseases such as arthritis. IκB kinase (IKK) is the converging point for the activation of NF-κB by a broad spectrum of inflammatory agonists and is thus a novel target for therapeutic intervention. We describe a small molecule, selective inhibitor of IKK-2, SC-514, which does not inhibit other IKK isoforms or other serine-threonine and tyrosine kinases. SC-514 inhibits the native IKK complex or recombinant human IKK-1/IKK-2 heterodimer and IKK-2 homodimer similarly. IKK-2 inhibition by SC-514 is selective, reversible, and competitive with ATP. SC-514 inhibits transcription of NF-κB-dependent genes in IL-1β-induced rheumatoid arthritis-derived synovial fibroblasts in a dose-dependent manner. When the mechanism of NF-κB activation was evaluated in the presence of this inhibitor, several interesting observations were found. First, SC-514 did not inhibit the phosphorylation and activation of the IKK complex. Second, there was a delay but not a complete blockade in IκBα phosphorylation and degradation; likewise there was a slightly slowed, decreased import of p65 into the nucleus and a faster export of p65 from the nucleus. Finally, both IκBα and p65 were comparable substrates for IKK-2, with similar Km and Kcat values, and SC-514 inhibited the phosphorylation of either substrate similarly. Thus, the effect of SC-514 on cytokine gene expression may be a combination of inhibiting IκBα phosphorylation/degradation, affecting NF-κB nuclear import/export as well as the phosphorylation and transactivation of p65.

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

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.

Regulation and Phenotype of an Innate Th1 Cell: Role of Cytokines and the p38 Kinase Pathway

We have explored the phenotype and regulation of Th1 cell activation by the cytokines IL-12 and IL-18. We demonstrate that these two cytokines selectively induce IFN-γ in a differentiated Th1 cell population through the previously described p38 mitogen-activated protein (MAP) kinase pathway. Using a highly selective p38 MAP kinase inhibitor, we demonstrate that it is possible to block IFN-γ induction from activated, differentiated Th1 cells via p38 MAP kinase without disrupting the activation and differentiation of naive T cells or the proliferation of naive or differentiated T cells. In addition, IL-12 and IL-18 provide an Ag and IL-2-independent survival signal to this uniquely differentiated Th1 cell population. We hypothesize that this Ag-independent survival of Th1 cells may participate in an innate inflammatory loop with monocytes at the sites of chronic inflammation. In addition, p38 MAP kinase inhibition of this cytokine-regulated pathway may be a unique mechanism to inhibit chronic inflammation without disruption of Ag-driven activation and function of naive T cells.

Kinetic Mechanisms of IκB-related Kinases (IKK) Inducible IKK and TBK-1 Differ from IKK-1/IKK-2 Heterodimer

Nuclear factor-κB activation depends on phosphorylation and degradation of its inhibitor protein, IκB. The phosphorylation of IκBα on Ser32 and Ser36 is initiated by an IκB kinase (IKK) complex that includes a catalytic heterodimer composed of IκB kinase 1 (IKK-1) and IκB kinase 2 (IKK-2) as well as a regulatory adaptor subunit, NF-κB essential modulator. Recently, two related IκB kinases, TBK-1 and IKK-i, have been described. TBK-1 and IKK-i show sequence and structural homology to IKK-1 and IKK-2. TBK-1 and IKK-i phosphorylate Ser36 of IκBα. We describe the kinetic mechanisms in terms of substrate and product inhibition of the recombinant human (rh) proteins, rhTBK-1, rhIKK-I, and rhIKK-1/rhIKK-2 heterodimers. The results indicate that although each of these enzymes exhibits a random sequential kinetic mechanism, the effect of the binding of one substrate on the affinity of the other substrate is significantly different. ATP has no effect on the binding of an IκBα peptide for the rhIKK-1/rhIKK-2 heterodimer (α = 0.99), whereas the binding of ATP decreased the affinity of the IκBα peptide for both rhTBK-1 (α = 10.16) and rhIKK-i (α = 62.28). Furthermore, the dissociation constants of ATP for rhTBK-1 and rhIKK-i are between the expected values for kinases, whereas the dissociation constants of the IκBα peptide for each IKK isoforms is unique with rhTBK-1 being the highest (K IκBα = 69.87 μm), followed by rhIKK-i (K IκBα = 5.47 μm) and rhIKK-1/rhIKK-2 heterodimers (K IκBα = 0.12 μm). Thus this family of IκB kinases has very unique kinetic properties.

Dual blockade of interleukin 1β and interleukin 17A reduces murine arthritis pathogenesis but also leads to spontaneous skin infections in non-human primates

Despite the efficacy of biologics for treatment of rheumatoid arthritis (RA), many patients show inadequate responses and likely require neutralization of multiple mediators. Neutralization of both interleukin (IL)-1β and IL-17A with monoclonal antibodies showed greater efficacy than either agent alone in a mouse arthritis model with cooperative inhibition of key inflammatory factors, IL-6, granulocyte colony-stimulating factor (G-CSF), and CXC chemokine ligand (CXCL)1. Given the potential clinical benefit in RA, we generated a human dual variable domain antibody Ig, ABBV-615, capable of simultaneous binding and neutralization of IL-1β and IL-17A. ABBV-615 was characterized and evaluated in cynomolgus monkeys for pharmacokinetics and toxicity to enable clinical development. ABBV-615 exhibited affinities (KD) of 12 and 3 pM on human IL-1β and IL-17A, respectively, and potencies (IC50) of 3 and 58 pM, respectively, as well as excellent drug-like properties. ABBV-615 pharmacokinetics in cynomolgus monkeys was dose proportional from 20 to 100 mg/kg with a mean half-life of 16 days. However, a 13-week repeat-dose toxicity study in cynomolgus monkeys revealed time-dependent spontaneous infections exclusively in skin at all doses tested and not historically seen with single-agent anti–IL-1α/β or anti–IL-17A. Consistent with reduced resistance to skin infections, IL-1β– and IL-17A–stimulated human keratinocytes demonstrate cooperative or compensatory production of key antibacterial and inflammatory mediators such as lipocalin-2, G-CSF, CXCL1, IL-8, tumor necrosis factor, and IL-6, which aid in defense against skin bacterial infections. These results illustrate the skin-specific antimicrobial mechanisms of IL-1β and IL-17A and highlight the importance of understanding unique combinatorial effects of biologic agents.