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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

Tumor necrosis factor ␣ (TNF␣) is a pro-inflammatory cytokine that controls the initiation and progression of inflammatory diseases such as rheumatoid arthritis. Tpl2 is a MAPKKK in the MAPK (i.e. ERK) pathway, and the Tpl2-MEK-ERK signaling pathway is activated by the pro-inflammatory mediators TNF␣, interleukin (IL)-1␤, and bacterial endotoxin (lipopolysaccharide (LPS)). Moreover, Tpl2 is required for TNF␣ expression. Thus, pharmacologic inhibition of Tpl2 should be a valid approach to therapeutic intervention in the pathogenesis of rheumatoid arthritis and other inflammatory diseases in humans. We have developed a series of highly selective and potent Tpl2 inhibitors, and in the present study we have used these inhibitors to demonstrate that the catalytic activity of Tpl2 is required for the LPS-induced activation of MEK and ERK in primary human monocytes. These inhibitors selectively target Tpl2 in these cells, and they block LPS-and IL-1␤-induced TNF␣ production in both primary human monocytes and human blood. In rheumatoid arthritis fibroblast-like synoviocytes these inhibitors block ERK activation, cyclooxygenase-2 expression, and the production of IL-6, IL-8, and prostaglandin E 2 , and the matrix metalloproteinases MMP-1 and MMP-3. Taken together, our results show that inhibition of Tpl2 in primary human cell types can decrease the production of TNF␣ and other pro-inflammatory mediators during inflammatory events, and they further support the notion that Tpl2 is an appropriate therapeutic target for rheumatoid arthritis and other human inflammatory diseases.

The optimization of a class of indole cPLA 2 alpha inhibitors is described herein. The importance of the substituent at C3 and the substitution pattern of the phenylmethane sulfonamide region are highlighted. Optimization of these regions led to the discovery of 111 (efipladib) and 121 (WAY-196025), which are shown to be potent, selective inhibitors of cPLA 2 alpha in a variety of isolated enzyme assays, cell based assays, and rat and human whole blood assays. The binding of these compounds has been further examined using isothermal titration calorimetry. Finally, these compounds have shown efficacy when dosed orally in multiple acute and chronic prostaglandin and leukotriene dependent in vivo models.

ABSTRACT:The metabolism of methoxychlor, a proestrogenic pesticide (endocrine disruptor), was investigated with cDNA expressed human cytochrome P450s and liver microsomes (HLM). In addition to 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethane (mono-OH-M), 1,1,1-trichloro-2, 2-bis(4-hydroxyphenyl)ethane (bis-OH-M), and 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(3, 4-dihydroxyphenyl)ethane (tris-OH-M), a new metabolite was identified as 1,1,1- Methoxychlor, 1 a biodegradable pesticide and a substitute for the banned DDT, has a relatively low toxicity and short half-life (Metcalf et al., 1971;Gardner and Bailey, 1975;Metcalf, 1976). Despite these favorable features of methoxychlor, there is considerable concern for exposure to methoxychlor because of its estrogenic activity (Bulger et al., 1978b;Ousterhout et al., 1981). Whereas in vitro methoxychlor has little or no affinity for the estrogen receptor (ER), in vivo methoxychlor exhibits pronounced estrogenic activity, indicating that methoxychlor per se is a proestrogen (Bulger et al., 1978a,b;Kupfer and Bulger, 1979;Bulger et al., 1985). Indeed, methoxychlor undergoes oxidative metabolism by hepatic cytochrome P450 forming metabolites with pronounced estrogenic activity (Bulger et al., 1978a,b;Bulger et al., 1985). Additionally, methoxychlor was found to elicit considerable endocrine and reproductive toxicity (Gray et al., 1988;Cummings and Gray, 1989;Cummings and Laskey, 1993). However, hitherto it has not been established whether the endocrine and reproductive toxicities are associated with methoxychlor per se or are primarily due to its metabolites. Based on such and other findings, methoxychlor was categorized as a prototype endocrine disruptor, and the nature of its toxicity has been under protracted intensive investigation (Chapin et al., 1997;Cummings, 1997;You et al., 2002). Interestingly, the mono-and bis-demethylated methoxychlor metabolites (mono-OH-M and bis-OH-M) exhibit opposing activities toward the ER␣ and ER␤ isoforms [i.e., both metabolites are agonists of ER␣, but antagonists of ER␤, as well as antagonists of the androgen receptor (Gaido et al., 2000)]. Additionally, it was reported that bis-OH-M activity differs considerably from that of estradiol-17␤ in affecting certain gene expression in mice ovaries (Waters et al., 2001), possibly explaining the distinctive but overlapping pathologies in trichloro-2-(4-methoxyphenyl)-2-(3, 4-dihydroxyphenyl)ethane (catechol-M; previously assumed to be ring-OH-M) and as a key metabolic intermediate. A novel metabolic route was proposed involving methoxychlor O-demethylation to mono-OH-M, followed by bifurcation of the pathway, both leading to the same final product tris-OH-M: pathway a, mono-OH-M is demethylated to bis-OH-M, followed by ortho-hydroxylation forming tris-OH-M and pathway b, mono-OH-M is ortho-hydroxylated forming catechol-M that is O-demethylatedThe project described was supported by an National Institutes of Health Grant ES00834 from the National Institute of Environmental Health Sci...

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