SUMMARY Increased intestinal permeability, i.e., barrier loss, is a common characteristic of many gastrointestinal and systemic disorders and has been hypothesized to be a key contributor to disease progression. Animal models of disease provide strong support for this hypothesis, but, due to the absence of drugs that reverse disease- associated permeability increases, the therapeutic potential of intestinal barrier restoration is untested. Myosin light chain kinase (MLCK) is a signaling node that, when activated, increases permeability. In inflammatory bowel disease (IBD) patient biopsies, MLCK activity is upregulated and correlates with disease severity. Recent studies have shown that inflammatory stimuli trigger recruitment of a specific MLCK isoform, MLCK1, to the perijunctional cytoskeleton and that this MLCK1 recruitment is required for barrier loss. Further, disruption of the MLCK1 interactome that directs recruitment is therapeutically superior to anti-TNF in experimental IBD. Therefore, the long-term goal of this program is to create the detailed understanding that will enable development of agents that target the MLCK1 interactome to restore barrier function and benefit IBD patients. The overall objectives of this application are to i) discover the molecular basis of MLCK1 interactome function and ii) apply this knowledge to block MLCK1 recruitment in experimental IBD. The central hypothesis is that prevention of MLCK1 interactome-dependent recruitment can restore the intestinal barrier. The rationale for this project is that elucidating the molecular mechanisms of MLCK1 interactome function and assessing the preclinical efficacy of targeting interactome components will create the scientific foundation needed for translation to the clinic. The central hypothesis will be tested via three specific aims: i) define mechanisms that regulate interactions between MLCK1 interactome components and identify sites that can be targeted to disrupt these interactions; ii) determine how, when, and where these interactions are modified by inflammatory signals and the potential of obstructing key interactions as a means to block MLCK1 recruitment and restore barrier function in intestinal stem cell models; and iii) characterize the impact of interfering with dynamic regulation of the MLCK1 interactome in preclinical mouse models and assess interactome staus in patient biopsies. This proposal is conceptually and technically innovative because it will define function and regulation of the newly-recognized MLCK1 interactome using sophisticated in vitro and in vivo models as well as patient biopsies. These studies are significant because they are expected to create the structural and mechanistic insight required for development of molecular therapies that modify unique MLCK1 interactome functions to prevent or reverse barrier loss in IBD. Ultimately, advances that build on this critical information will create a foundation for development of safe, non-immuno...