Environmental stress and inflammatory cytokines activate the transcription factor NFKB and a cascade of protein kinases that result in the activation of the Stress Activated Protein Kinase (SAPK, also known as JNK). While some upstream kinases that activate SAPK are characterized, intermediate events that transmit stress signals between the stress agent receptor and the protein kinase cascade are unclear. Reactive oxygen species (ROS) are thought to be involved because high levels of thiol-containing chemicals can block activation of NFKB and SAPK. The investigators have completed a two hybrid interaction screen to identify proteins that can interact with the SAPK activator MEKK1, which our laboratory identified as a proximate activator of the SAPK protein kinase cascade. Surprisingly, a NAD(P)H-quinone reductase termed NQO1 (DT-diaphorase) was identified as a MEKK1 binding protein. NQO1 is inhibited by compounds (generally quinones) that may serve as competitive substrate inhibitors. Their preliminary data also show that these agents are strongly inhibitory of the activation of SAPK and activation of NFKB, and have termed them Stress Inhibitory Compounds (SICs). These agents are approximately 1000 fold more effective towards inhibiting SAPK and NFKB than are thiol-containing agents. Initial aims in this application are to develop improved SICs with still lower IC50s through rational drug design (aim 1). Secondly, using a genetic selection in yeast in which SICs prevent survival in situations of stress, yeast and human genes that overcome this lethal combination of SIC and stress will be isolated (aim 2). These represent potential targets of SIC action. Inflammation underlies the pathogenesis of environmental-induced diseases including those broadly-termed hypersensitivity pneumonitis which is caused by a wide variety of agents including grain dusts and the lipopolysaccharide (LPS) contaminating these dusts. The inflammatory response to these environmental stimuli is accompanied by secretion of cytokines which in turn may strongly activate SAPK and NFKB. The investigators preliminary data show that SICs inhibit LPS-mediated activation of both of these stress pathways in human alveolar macrophages, a major source of inflammatory and injurious mediators in the lung during development of hypersensitivity pneumonitis. Thus, the investigators hypothesize that SICs may represent a novel means of interrupting the pathophysiologic processes leading to the restrictive lung disease characteristic of hypersensitivity pneumonitis, regardless of the environmental stimulus. They will employ both in vitro models using human alveolar macrophages (aim 3) and mouse models of LPS and grain dust-induced lung damage (aim 4) to test the ability of SICs to interrupt the biochemical and biological consequences of these agents.
