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We have developed novel orally active quinazoline analogues as inhibitors of AP-1 and NF-kappaB mediated transcriptional activation. Among the derivatives prepared, 1-[2-(2-thienyl)quinazolin-4-ylamino]-3-methyl-3-pyrroline-2,5-dione (10) showed significant activity in an adjuvant-induced arthritis rat model by reducing the swelling by 65% in the non-injected foot. The synthesis, structure-activity relationship, and in vivo activity are described.

NF-kappa B plays a key role in the production of cytokines in inflammatory diseases. The effects of a novel T cell-specific NF-kappa B inhibitor, SP100030, were evaluated in cultured Jurkat cells and in murine collagen-induced arthritis (CIA). Chemical libraries were screened for NF-kappa B-inhibitory activity. SP100030, a compound identified in this process, inhibited NF-kappa B activation in PMA/PHA-activated Jurkat cells by EMSA at a concentration of 1 microM. Jurkat cells and the monocytic cell line THP-1 were transfected with an NF-kappa B promotor/luciferase construct and activated. SP100030 inhibited luciferase production in the Jurkat cells (IC50 = 30 nM). ELISA and RT-PCR confirmed that IL-2, IL-8, and TNF-alpha production by activated Jurkat and other T cell lines were inhibited by SP100030. However, cytokine expression was not blocked by the compound in THP-1 cells, fibroblasts, endothelial cells, or epithelial cells. Subsequently, DBA/1J mice were immunized with type II collagen. Treatment with SP100030 (10 mg/kg/day i.p. beginning on day 21) significantly decreased arthritis severity from onset of clinical signs to the end of the study on day 34 (arthritis score, 5.6 +/- 1.7 for SP100030 and 9.8 +/- 1.5 for control; p < 0.001). Histologic evaluation demonstrated a trend toward improvement in SP100030-treated animals. EMSA of arthritic mouse ankles in CIA showed that synovial NF-kappa B binding was suppressed in the SP100030-treated mice. SP100030 inhibits NF-kappa B activation in T cells, resulting in reduced NF-kappa B-regulated gene expression and decreased CIA. Its selectivity for T cells could provide potent immunosuppression with less toxicity than other NF-kappa B inhibitors.

The product of the proto-oncogene Jun inhibits myogenesis. Constitutive expression of Jun in myoblasts interferes with the expression and the function of MyoD protein. In transient transfection assays Jun inhibits transactivation of the MyoD promoter, the muscle creatine kinase enhancer, and a reporter gene linked to MyoD DNA-binding sites. Conversely, MyoD suppresses the transactivation by Jun of genes linked to an AP-1 site. We demonstrate that both in vivo and in vitro MyoD and Jun proteins physically interact. Mutational analysis suggests that this interaction occurs via the leucine zipper domain of Jun and the helix-loop-helix region of MyoD.

A total of 28 L-rhamnose-negative mutants in Salmonella typhimurium LT2 were all linked by P22 transduction and were classified into five groups on the basis of genetic and biochemical experiments. Deletion mapping showed that the gene order was rhaD rhaA rhaB rhaC rhaT . rhaA mutants lacked an inducible L-rhamnose isomerase, rhaB mutants lacked an inducible L- rhamnulokinase , and rhaD mutants were probably defective in L- rhamnulose -1-phosphate aldolase. Mutants that were unable to accumulate L-[14C]rhamnose but could grow on 1% L-rhamnose were designated rhaT to indicate a defect in L-rhamnose transport. Genetic evidence supports the hypothesis that the rhaC gene is a positive regulator of rha gene expression. (i) Pleiotropically negative mutants in the rhaC gene were isolated at a high frequency. (ii) Mutants containing an insertion or deletion within the rhaC gene had a pleiotropically negative phenotype. (iii) Complementation tests indicated that rhaC + was dominant to rhaC -. (iv) Rha+ revertants of deletion and Tn10 insertion mutations in the rhaC gene were isolated.

An NK-like cell line (YTN10 cells) was stimulated with IL-1, and the IL-2R, as detected by flow cytometry analysis using fluorescein-labeled anti-IL-2R (Tac) antibody, increased displaying a dose-response curve in the shape of an inverted "U." This was associated with increases in 1) the number of high affinity binding sites for IL-2 (Kd approximately 45 pM) which increased from 1270 to 4220 sites per cell, and 2) the number of binding sites for anti-Tac antibody, which increased from 999 to 6763 sites per cell. They were both detected by using radiolabeled ligand-binding methods which are one to three orders of magnitude more sensitive than flow cytometry analysis. No binding sites with low affinity for IL-2 could be detected on untreated or treated cells. However, a change was noted in the number of IL-2 binding sites with intermediate affinity (Kd approximately 1000 pM), which increased from 54,000 to 67,000 sites per cell. This binding affinity is equivalent to that of the p70 IL-2-binding protein detected on YT cells and on large granular lymphocytes by other investigators. This suggests that the increase in the Tac peptide was the limiting factor in the combination of the Tac and p70 components to form the high affinity IL-2R. The corollary is that all new Tac molecules were incorporated into these R. The IL-1 treatment also induced an increase in production of Tac mRNA as detected by Northern blots. Comparison of the relative amounts of Tac mRNA by dot blots verified the quantitative difference between treated and untreated cells. These data demonstrate that the IL-1-induced positive regulation of the Tac protein and high affinity IL-2R on YTN10 cells may operate at the level of gene transcription.