IL-6 is a major causative factor of inflammatory disease. Although IL-6 and its signaling pathways are promising targets, orally available small-molecule drugs specific for IL-6 have not been developed. To discover IL-6 antagonists, we screened our in-house chemical library and identified LMT-28, a novel synthetic compound, as a candidate IL-6 blocker. The activity, mechanism of action, and direct molecular target of LMT-28 were investigated. A reporter gene assay showed that LMT-28 suppressed activation of STAT3 induced by IL-6, but not activation induced by leukemia inhibitory factor. In addition, LMT-28 downregulated IL-6–stimulated phosphorylation of STAT3, gp130, and JAK2 protein and substantially inhibited IL-6–dependent TF-1 cell proliferation. LMT-28 antagonized IL-6–induced TNF-α production in vivo. In pathologic models, oral administration of LMT-28 alleviated collagen-induced arthritis and acute pancreatitis in mice. Based on the observation of upstream IL-6 signal inhibition by LMT-28, we hypothesized IL-6, IL-6Rα, or gp130 to be putative molecular targets. We subsequently demonstrated direct interaction of LMT-28 with gp130 and specific reduction of IL-6/IL-6Rα complex binding to gp130 in the presence of LMT-28, which was measured by surface plasmon resonance analysis. Taken together, our data suggest that LMT-28 is a novel synthetic IL-6 inhibitor that functions through direct binding to gp130.

BACKGROUND AND PURPOSEHepatitis C virus (HCV) infection is responsible for various chronic inflammatory liver diseases. Here, we have identified a naturally occurring compound with anti-HCV activity and have elucidated its mode of antiviral action. EXPERIMENTAL APPROACHLuciferase reporter and real-time RT-PCR assays were used to measure HCV replication. Western blot, fluorescence-labelled HCV replicons and infectious clones were employed to quantitate expression levels of viral proteins. Resistant HCV mutant mapping, in vitro NS3 protease, helicase, NS5B polymerase and drug affinity responsive target stability assays were also used to study the antiviral mechanism. KEY RESULTSA resveratrol tetramer, vitisin B from grapevine root extract showed high potency against HCV replication (EC 50 = 6 nM) with relatively low cytotoxicity (EC 50 >10 μM). Combined treatment of vitisin B with an NS5B polymerase inhibitor (sofosbuvir) exhibited a synergistic or at least additive antiviral activity. Analysis of a number of vitisin B-resistant HCV variants suggested an NS3 helicase as its potential target. We confirmed a direct binding between vitisin B and a purified NS3 helicase in vitro. Vitisin B was a potent inhibitor of a HCV NS3 helicase (IC 50 = 3 nM). In vivo, Finally, we observed a preferred tissue distribution of vitisin B in the liver after i.p. injection in rats, at clinically attainable concentrations. CONCLUSION AND IMPLICATIONSVitisin B is one of the most potent HCV helicase inhibitors identified so far. Vitisin B is thus a prime candidate to be developed as the first HCV drug derived from natural products.

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The Her-2/neu oncogene, which encodes a growth factor receptor, was implicated in the malignancy of human adenocarcinomas. Antibodies directed to this molecule have been previously shown to have an antitumor effect in vivo. In an attempt to understand the mechanisms of the antitumor activity, we generated 2 monoclonal antibodies (mAbs), HRO G1 and HRT G1, that recognize different epitopes on Her-2/neu. Both of the mAbs bound HER2/neu on the tumor surface, resulting in phosphorylation of HER2/neu. We also generated IgG2a and IgG2b mAbs from these 2 mAbs, respectively. The results of in vitro studies showed that these anti-Her-2/neu mAbs could not inhibit the growth of the tumor cells that express Her-2/neu molecules by themselves. However, in an antibody-dependent cellular cytotoxicity study using mouse splenocytes as effector cells, HRT mAbs had antitumor activities superior to those of HRO mAbs, indicating that the epitope specificity may also partake in antibody-dependent cellular cytotoxicity with antibody isotype. In a complement-dependent cytotoxicity study, the IgG2a and IgG2b mAbs showed stronger effects than IgG1 isotype mAbs irrespective of the epitope specificities. The results of in vivo studies also showed that HRT mAbs had superior antitumor activity to those of HRO mAbs. The antitumor activity was most prominent in the HRT G2b isotype among HRT mAbs. HRT G1 also showed a moderate antitumor effect, while HRT G2a showed only slight inhibition effect. These data indicate that both the epitope specificity and the differences in Fc region of mAbs could play important roles in the antitumor activities. © 2002 Wiley-Liss, Inc. Key words: Her-2/neu; monoclonal antibody; isotype; epitope; antitumor effectThe Her-2/neu gene encodes a M r 185 kDa transmembrane protein that is a member of the type I family of growth factor receptors. 1,2 Amplification of this gene results in overexpression of the 185-kd encoded receptor tyrosine kinase, which is homologous to the EGF 3 receptor. 3-5 However, unlike the EGF receptor, which binds many known ligands, no direct ligand of Her-2/neu has been reported. 6 The Her-2/neu protein was found to be amplified and overexpressed in several types of human adenocarcinomas, especially in tumors of the breast and the ovary. 5,7,8 The overexpression was correlated with short time to relapse and poor survival of breast cancer patients, 9 -11 suggesting that Her-2/neu overexpression likely plays a critical role in the development of human cancers. Several lines of evidence also support a direct role of Her-2/neu in the pathogenesis and clinical aggressiveness of Her-2/neu-expressing tumors. 12 In fact, a large number of published studies demonstrated that Her-2/neu overexpression is a cause of human cancer and not just a consequence. Therefore, Her-2/neu oncogene is an excellent target for development of therapeutic agents specific for Her-2/neu overexpressing human cancers.A number of approaches have been used to therapeutically target Her-2/neu-overexpressing cancers. A common appr...

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