Autoimmune diseases result from a breakdown of self-tolerance. Disease progression involves several key steps, including the inappropriate activation of autoreactive lymphocytes followed by the infiltration of pathogenic effectors T cells into the target tissues. Our recent studies have focused on a mouse model of multi- organ autoimmune disease that results from the absence of the costimulatory molecule, CTLA-4 (i.e., Ctla4-/- mice). In this model, mice succumb to a rapid and fatal disease that results from massive T cell activation, infiltration into vital organs, and the subsequent failure of those organs. We have found that the Tec family tyrosine kinase ITK plays a critical role in the process of autoreactive T cell migration into tissues in this multi- organ autoimmune disease. Thus, Itk-/-Ctla4-/- mice show unprecedented T cell activation and proliferation, but are protected from the lethal autoimmunity of Ctla4-/- mice due to a failure of these activated effectors T cells to accumulate in non-lymphoid tissues. In addition, we can prevent the onset of severe autoimmune disease in Ctla4-/- mice by treatment with a small molecule ITK inhibitor. ITK is a well-characterized signaling protein that is activated by TCR, CD28, and chemokine receptor stimulation;in turn, ITK activates phospholipase-Cg and induces actin polymerization. These findings indicate that ITK signaling in effectors T cells is critical in regulating T cell infiltration into tissues, and more importantly, that ITK is essential for the pathogenesis of autoimmune T cells. In this project, we propose to establish and validate a biochemical assay to screen for novel small molecule inhibitors of ITK. Although two groups have previously reported the identification of ITK inhibitors, these inhibitors are of relatively low potency and have poor pharmacokinetics in vivo. Moreover, the screens that produced these compounds used the isolated ITK kinase domain and so yielded only ATP-site directed inhibitors. Based on our in depth biochemical investigations of full length ITK and our recent description of a remote ITK substrate docking mechanism, we have begun to establish a greatly improved ITK in vitro kinase assay, in which the Km of ITK for its substrate is >15-fold higher than in previously-established assays. Use of this novel assay will provide a platform for identifying inhibitors that are efficacious at lower concentrations and with higher selectivity for ITK. In the first aim, we will optimize the ITK in vitro kinase assay to maximize reproducibility and to determine assay conditions amenable to HTS. In the second aim, we will establish cell-based and whole animal assays for ITK inhibitors as secondary and tertiary screens, including counter-screens for ITK inhibition. Our overall goal is to establish a robust high-throughput screen to identify novel, selective, and high affinity small molecule inhibitors of ITK, and ultimately, to assess the efficacy of these inhibitors in animal models of organ-specific autoimmune diseases, as well as in the inhibition of human T cell migration and diapedesis.

PUBLIC HEALTH RELEVANCE: Autoimmune diseases occur when an individual's immune system attacks his/her own cells and organs. We have identified an enzyme that plays an essential role in this disease process. This proposal aims to develop a screen for inhibitors of this enzyme, which could be used to prevent the onset of autoimmune processes in susceptible individuals and to treat and cure patients with these diseases.

R21NS071548

2010-07-01

2012-06-30