MASSACHUSETTS INSTITUTE OF TECHNOLOGY
Our long-term objective for this proposed research is to discover novel treatments for red cell disorders with hemoglobinopathy. In this supplement proposal, we will accelerate and extend the research proposed in the parent grant for the screening small chemical libraries for compounds that modulate HRI stress response pathway and reduce apoptosis of beta-thalassemic erythroid precursors. Thalassemia is the most common monogenic disease in the world, and is emerging as a major health and economic burden in the world. Hem-regulated eIF2 alpha kinase (HRI) was initially discovered to inhibit general protein synthesis in heme-deficiency of erythroid precursors. Thus, HRI balances heme and globin synthesis in erythroid precursors to prevent accumulation of excess heme or globin, which are toxic to cells. However, the function of HRI is not limited to heme-deficiency. HRI is also activated by sodium arsenite-induced oxidative stress, osmotic shock and heat shock. Most importantly, HRI is the principal eIF2 alpha kinase responsible for protection of erythroid cells against heme-deficiency, oxidative stress and heat shock. Our laboratory has shown that HRI is necessary to reduce the phenotypic severity not only in iron-deficiency anemia, but also in beta-thalassemia. HRI mediates this protection in part by phosphorylation of eIF2 alpha and inhibition of protein synthesis including globin synthesis to prevent excessive accumulation of globin aggregates. Recently, our laboratory has found that activation of HRI upon sodium arsenite exposure also induces an oxidative stress signaling pathway in nucleated erythroid precursors to mitigate toxicity of arsenite. Additionally, this HRI stress signaling pathway is also activated during erythroid differentiation and is necessary for erythroid differentiation under stress conditions of iron deficiency and beta-thalassemia intermedia. Recent studies have demonstrated that inhibition of erythroid differentiation is the major underlying cause of ineffective erythroipoiesis in beta-thalassemia. Thus, HRI and the components of its downstream signaling pathways may be potential pharmaceutical targets for the development of novel treatments of severe thalassemia. Towards this end, 12,500 compounds from libraries of small chemicals were screened for protection against arsenite toxicity in erythroid precursors. Thirteen lead compounds have been identified. The specific aims of this proposal are (1) To investigate the efficacy of these 13 small molecules for reducing severity in mouse model of beta-thalssemia. (2) To investigate the molecular action of these lead compounds in protection against oxidative stress and beta-thalssemia. Techniques that will be employed are a cell culture system for erythroid differentiation ex vivo, fluorescent activated cell sorting analysis of oxidative stress and erythroid differentiation, protein and RNA expression analyses of the HRI signaling pathway. The outcome of these proposed studies may lead to the discovery of novel compounds for treatments of not only thalassemia but also red cell disorders generally.