Identifying a specific small molecule to reverse intestinal barrier loss

A new study published in Nature Medicine reports that a small molecule targeting myosin light chain kinase (MLCK)1 improves colitis-associated intestinal barrier function in mice.

Colonic biopsy sample from a patient with IBD. Cell nuclei are shown in blue, E-cadherin is shown in green and the tight junction protein ZO-1 is shown in red. Image courtesy of M. L. D. M. Ong and J. Turner, Brigham and Women’s Hospital, USA.

An essential function of the intestinal epithelium is to serve as a barrier between the contents of the gut lumen and host tissue. This monolayer of cells, connected by tight junctions (TJs), balances protective functions with the need to allow passage of nutrients and water and regulate intestinal immune homeostasis. Importantly, impaired barrier function (increased intestinal permeability) is a key feature of intestinal diseases — for instance, increased permeability during remission predicts relapse in patients with Crohn’s disease.

Spurred by these observations, Jerrold Turner and colleagues at Brigham and Women’s Hospital, Boston, USA, began investigating the physiological mechanisms regulating intestinal epithelial barrier function. Over the course of more than two decades, they and other groups demonstrated a crucial role for MLCK in regulating TJ function in physiological and pathophysiological settings. However, the translation of these findings was hindered by the toxicity of systemic inhibitors of MLCK enzymatic activity. In addition to regulating TJ function, MLCK also has critical roles in smooth muscle, and inhibition of the MLCK catalytic domain results in hypotension, loss of intestinal motility and death.

As first reported in the new paper, a key breakthrough came when the Turner group observed that inflammatory stimuli promote recruitment of the MLCK splice variant MLCK1, but not MLCK2, to TJs. This finding opened the door to more targeted approaches. “After realizing that MLCK1 was recruited to the TJ but that MLCK2 was not, we decided to target the structural domain that represents the only difference between these two splice variants, IgCAM3,” explains Turner. “We used the structure of this domain and an in silico docking strategy to screen a large database of small, drug-like molecules and identify a few dozen to test in biological systems.”

One small-molecule compound was identified that prevented MLCK-dependent increases in intestinal epithelial permeability, without inhibiting enzymatic activity or altering smooth muscle function. This compound, termed divertin by the authors, was confirmed to bind MLCK1 IgCAM3 at or near the predicted binding site. In a series of in vitro assays, the team demonstrated the mechanism of action of divertin. “Rather than inhibit the enzymatic activity of myosin light chain kinase, we prevented it from getting to the subcellular site of action,” says Turner. “As a result, MLCK-mediated TJ dysregulation was blocked, but other MLCK functions in epithelial cells and non-epithelial cells, such as smooth muscle cells, remained intact.”

Next, the researchers investigated the in vivo effects of divertin. Using in situ perfused mouse jejunal segments, the team showed that loss of intestinal barrier function induced by intraperitoneal injection of TNF, an archetypal disruptor of epithelial TJs, was prevented when divertin was administered to the jejunal lumen. Analysis of this tissue revealed the same mechanism of action as in the in vitro experiments, and experiments in mucosal biopsy samples taken from healthy individuals confirmed that divertin also prevents MLCK1 recruitment to TJs in humans.

Finally, Turner and colleagues assessed the effects of divertin in different mouse models of colitis. “The colitis models show that it was remarkably effective in both treating early-stage experimental inflammatory bowel disease and, in a model that recapitulates disease reactivation from remission, in preventing colitis progression,” says Turner. Importantly, the doses of divertin used did not induce weight loss or behaviour changes, and there was no evidence of histopathological toxicity in intestinal tissue samples.

“the doses of divertin used did not induce weight loss or behaviour changes”

“The structure and relatively low affinity of divertin for IgCAM3 suggest that it will not become a drug for human use,” Turner acknowledges. “However, it is a proof-of-concept molecule that can also serve as a tool compound for development of agents with similar biological activities that are more suitable for use as therapeutics.” The group is now working to define the mechanisms of MLCK1 recruitment to TJs, which could uncover further targets for future drugs.


Original article

  1. Graham, W. V. et al. Intracellular MLCK1 diversion reverses barrier loss to restore mucosal homeostasis. Nat. Med. 25, 690–700 (2019)

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Correspondence to Hugh Thomas.

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Thomas, H. Identifying a specific small molecule to reverse intestinal barrier loss. Nat Rev Gastroenterol Hepatol 16, 325 (2019).

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