Identification and Characterization of a Novel Class of c-Jun N-terminal Kinase Inhibitors

Abstract

In efforts to identify novel small molecules with antiinflammatory properties, we discovered a unique series of tetracyclic indenoquinoxaline derivatives that inhibited lipopolysaccharide (LPS)-induced nuclear factor-B/activating protein 1 activation. Compound IQ-1 (11H-indeno[1,2-b]quinoxalin-11-one oxime) was found to be a potent, noncytotoxic inhibitor of pro-inflammatory cytokine [interleukin (IL)-1␣, IL-1␤, IL-6, IL-10, tumor necrosis factor (TNF)-␣, interferon-␥, and granulocyte-macrophage colony-stimulating factor] and nitric oxide production by human and murine monocyte/macrophages. Three additional potent inhibitors of cytokine production were identified through further screening of IQ-1 analogs. The sodium salt of IQ-1 inhibited LPS-induced TNF-␣ and IL-6 production in MonoMac-6 cells with IC 50 values of 0.25 and 0.61 M, respectively. Screening of 131 protein kinases revealed that derivative IQ-3 [11H-indeno[1,2-b]quinoxalin-11-one-O-(2-furoyl)oxime]was a specific inhibitor of the c-Jun N-terminal kinase (JNK) family, with preference for JNK3. This compound, as well as IQ-1 and three additional oxime indenoquinoxalines, were found to be high-affinity JNK inhibitors with nanomolar binding affinity and ability to inhibit c-Jun phosphorylation. Furthermore, docking studies showed that hydrogen bonding interactions of the active indenoquinoxalines with Asn152, Gln155, and Met149 of JNK3 played an important role in enzyme binding activity. Finally, we showed that the sodium salt of IQ-1 had favorable pharmacokinetics and inhibited the ovalbumin-induced CD4 ϩ T-cell immune response in a murine delayed-type hypersensitivity model in vivo. We conclude that compounds with an indenoquinoxaline nucleus can serve as specific small-molecule modulators for mechanistic studies of JNKs as well as a potential leads for the development of anti-inflammatory drugs.

Anti-Inflammatory Effects and Joint Protection in Collagen-Induced Arthritis after Treatment with IQ-1S, a Selective c-Jun N-Terminal Kinase Inhibitor

“…We also confirmed that IQ-1S inhibited c-Jun phosphorylation in FLSs (Fig. 3C), which confirms our previous observation in human MonoMac-6 cells (Schepetkin et al, 2012). Together, these data suggest that IQ-1S may protect joints in part by decreasing MMP1/MMP3 expression.…”

“…We previously reported that the binding affinities (K d ) of IQ-1S toward JNK1, JNK2, and JNK3 were 390, 360, and 87 nM, respectively (Schepetkin et al, 2012). However, a detailed kinase…”

“…JNK inhibitor IQ-1S was synthesized as previously described (Schepetkin et al, 2012). Ketone derivative 11H-indeno [1,2-b]quinoxalin-11-one (compound IQ-18; compound designation as in our previous publication; Schepetkin et al, 2012) was purchased from Sigma-Aldrich (St. Louis, MO). For in vitro studies, IQ-1S was dissolved in dimethylsulfoxide (DMSO) and diluted into the desired buffer or culture media.…”

“…We previously determined that the serum IQ-1S concentration was 7.4 mM Â h 21 after intraperitoneal administration of 30 mg/kg IQ-1S in mice (Schepetkin et al, 2012). On the basis of these pharmacokinetic data and the observation that at least 4 mM IQ-1S was required for in vitro activity in the FLS assay, we used 30 mg/kg i.p.…”

“…We recently identified IQ-1 (11H-indeno [1,2-b]quinoxalin-11-one oxime) using high-throughput screening and demonstrated that this compound and its oxime analogs inhibited JNK enzymatic activity and, consequently, proinflammatory cytokine production by different murine and human cell systems (Schepetkin et al, 2012). In this study, we investigated the effect of the IQ-1S (11H-indeno [1,2-b]quinoxalin-11-one oxime sodium salt) on mouse collagen-induced arthritis (CIA), a common animal model of RA.…”