Scientific Abstract
Principal Investigator: Jerrold R. Turner, M.D., Ph.D.
Applicant Organization: The University of Chicago (Illinois, U.S.A.)
Project Title: Epithelial myosin light chain kinase trafficking: a therapeutic target in inflammatory bowel disease
Award Period: May 1, 2009 – May 31, 2012
Increased intestinal permeability is present in patients with Crohn’s disease (CD) and a subset of their healthy first-degree relatives. The resulting barrier dysfunction is involved in disease pathogenesis. We have shown that a key physiological regulator of the intestinal epithelial barrier, myosin light chain kinase (MLCK), mediates TNF-induced barrier dysfunction and that MLCK inactivated in human and experimental inflammatory bowel disease (IBD). Our recent data demonstrate that direct in vivo activation of MLCK within intestinal epithelia causes barrier defects comparable to those reported in human IBD patients and their relatives, that these barrier defects trigger both pro-and anti-inflammatory mucosal immune responses, and that barrier defects accelerate onset and progression of experimental IBD. Conversely, our preliminary data suggest that correction MLCK-dependent barrier defects delays onset and lessens severity of experimental IBD. While we have developed a highly-specific enzymatic inhibitor of MLCK that prevents acute, immune-mediated diarrheal disease in mice, we have also demonstrated that this agent interferes with in vitro closure of model epithelial intestinal wounds. Moreover, the MLCK catalytic domain, against which the inhibitor is targeted, is conserved in all smooth muscle and non-muscle MLCK isoforms. Thus, for MLCK to be a viable therapeutic target in IBD, an alternative, non-enzymatic approach to MLCK inhibition is needed. Our preliminary data demonstrate that a specific long MLCK splice variant, MLCK1, is the predominant form at the tight junction and is trafficked to the perijunctional actomyosin ring and tight junction in response to acute TNF treatment, both in vitro and in vivo, in animal models of chronic disease, and in specimens from IBD patients. We have now identified an immunoglobulin-like domain that is uniquely present within MLCK1 and responsible for directing MLCK1to the perijunctional actomyosin ring and identified small molecules that bind to the domain. The preliminary data suggest that these drug-like small molecules are able to: i) restore acute TNF-induced barrier loss in vitro, ii) prevent acute TNF-induced MLC phosphorylation in vitro and in vivo, and iii) prevent TNF-induced occludin endocytosis, barrier loss, and acute diarrhea in vivo; all without inhibiting MLCK enzymatic activity.
The aim of this proposal is to determine the ability of identified drugs to delay or prevent progression of experimental IBD in vivo. These data are expected to accelerate development of a novel class of non-immunosuppressive agents that may be useful, as part of multi-agent therapy, in maintaining remission.