Abstract

The induction of human inducible nitric-oxide synthase (iNOS) expression depends (among other factors) on activation of the signal transducer and activator of transcription 1 (STAT1) pathway. Therefore, the STAT1 pathway may be an appropriate target for the development of inhibitors of iNOS expression. HeLa S3 cells transiently transfected with a γ-activated site (GAS)/interferon-stimulated response element-driven reporter gene construct were used as the primary screening system. Using this system, three novel inhibitors of interferon-γ-dependent gene expression, namely, sporogen, S14-95, andS-curvularin, were isolated from differentPenicillium species. These three compounds also inhibited cytokine-induced, GAS-dependent reporter gene expression in stably transfected human A549/8-pGASLuc cells, confirming the data obtained with the above-mentioned screening system. Furthermore, in A549/8 cells, sporogen, S14-95, and S-curvularin inhibited cytokine-induced activity of the human iNOS promoter [a 16-kilobase (kb) fragment in stably transfected A549/8-pNOS2(16)Luc cells], cytokine-induced iNOS mRNA expression, and cytokine-induced nitric oxide (NO) production in a concentration-dependent manner. The proliferation of A549/8 cells, and the activity of the human eNOS promoter (a 3.5-kb fragment in stably transfected ECV-pNOS III-Hu-3500-Luc cells), were only influenced marginally by the three compounds. Sporogen, S14-95, and S-curvularin also inhibited cytokine-induced activation of STAT1α in A549/8 cells. In conclusion, sporogen, S14-95, and S-curvularin represent new transcriptionally based inhibitors of iNOS-dependent NO production, acting on the Janus tyrosine kinase-STAT pathway. These compounds may represent lead structures for the development of drugs inhibiting iNOS-dependent overproduction of NO in pathophysiological situations.