Epithelial barrier loss is a driver of intestinal and systemic diseases. Myosin light chain kinase (MLCK) is a key effector of barrier dysfunction and a potential therapeutic target, but enzymatic inhibition has unacceptable toxicity. Here, we show that a unique domain within the MLCK splice variant MLCK1 directs perijunctional actomyosin ring (PAMR) recruitment. Using the domain structure and multiple screens, we identify a domain-binding small molecule (divertin) that blocks MLCK1 recruitment without inhibiting enzymatic function. Divertin blocks acute, tumor necrosis factor (TNF)-induced MLCK1 recruitment as well as downstream myosin light chain (MLC) phosphorylation, barrier loss, and diarrhea in vitro and in vivo. Divertin corrects barrier dysfunction and prevents disease development and progression in experimental inflammatory bowel disease. Beyond applications of divertin in gastrointestinal disease, this general approach to enzymatic inhibition by preventing access to specific subcellular sites provides a new paradigm for safely and precisely targeting individual properties of enzymes with multiple functions.

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All requests for raw and analyzed data and materials are promptly reviewed to verify if the request is subject to any intellectual property or confidentiality obligations. Human participants were de-identified and no further data are available. Any data and materials that can be shared will be released via a Material Transfer Agreement. The crystal structure data are available as Protein Data Bank code: 6C6M (https://www.rcsb.org/structure/6C6M).

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This study was supported by National Institutes of Health (NIH) grant nos. R01DK61931 (J.R.T.), R01DK068271 (J.R.T.), R24DK099803 (J.R.T.), R01GM081030 (L.W.M.), R01AG048793 (S.C.M.), P30CA014599 (University of Chicago Comprehensive Cancer Center), P30DK034854 (the Harvard Digestive Disease Center), and T32HL007237 (W.V.G., A.M.M.). Work was also supported by the Broad Medical Research Program (IBD-022, to J.R.T.), the Department of Defense (W81XWH-09-1-0341, to J.R.T.), a Catalyst Award from the Chicago Biomedical Consortium (J.R.T., L.W.M.), the National Natural Science Foundation of China (81470804 and 31401229 to W.H.). The Berkeley Center for Structural Biology is supported in part by the NIH, National Institute of General Medical Sciences, and the Howard Hughes Medical Institute. The Advanced Light Source is a Department of Energy Office of Science User Facility under contract no. DE-AC02-05CH11231.

Author information

Author notes

  1. These authors contributed equally: W. Vallen Graham and Weiqi He.


  1. Department of Pathology, University of Chicago, Chicago, IL, USA

    • W. Vallen Graham
    • , Weiqi He
    • , Amanda M. Marchiando
    • , Juanmin Zha
    • , Gurminder Singh
    • , Yitang Wang
    • , Jingshing Wu
    • , Harry J. Rosenberg
    • , Yingmin Wang
    • , Stephen C. Meredith
    •  & Jerrold R. Turner
  2. Laboratory of Chemical Biology & Signal Transduction, The Rockefeller University, New York, NY, USA

    • W. Vallen Graham
  3. Jiangsu Key Laboratory of Neuropsychiatric Diseases and Cambridge-Suda Genomic Resource Center, Soochow University, and Department of Oncology, The First Affiliated Hospital of Soochow University, Suzhou, China

    • Weiqi He
    • , Juanmin Zha
    • , Hua-Shan Li
    •  & Zhi-Hui Jiang
  4. Laboratory of Mucosal Barrier Pathobiology, Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA

    • Gurminder Singh
    • , Ma. Lora Drizella M. Ong
    • , Wangsun Choi
    •  & Jerrold R. Turner
  5. Division of Gastroenterology, Hepatology and Nutrition, Boston Children’s Hospital and Harvard Medical School, Boston, MA, USA

    • Amlan Biswas
    •  & Scott B. Snapper
  6. Vertex Pharmaceuticals, Boston, MA, USA

    • Harmon Zuccola
    •  & James Griffith
  7. Department of Pathology, Immunology and Laboratory Medicine, University of Florida, College of Medicine, Gainesville, FL, USA

    • David Ostrov
  8. Department of Chemistry, University of Illinois at Chicago, Chicago, IL, USA

    • Lawrence W. Miller


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J.R.T. conceptualized the study. W.V.G., W.H., A.M.M., J.Z., G.S., H.-S.L., A.B., M.L.D.M.O., Z.-H.J., W.C., H.Z., Yitang Wang, J.G., J.W., H.J.R., Yingmin Wang, and J.R.T. carried out the investigations. S.B.S., D.O., S.C.M., L.W.M., and J.R.T. managed the resources. H.Z., J.G., and D.O. managed the software. W.V.G., W.H., W.C., and J.R.T. carried out the visualization. W.V.G. and, J.R.T. wrote the original manuscript draft. All authors reviewed and edited the draft. J.R.T. and W.H. acquired the funding. These pairs of authors contributed equally: W.V.G. and W.H., and A.M.M. and J.Z.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Jerrold R. Turner.

Extended data

  1. Extended Data Fig. 1 IgCAM3 drug-binding pocket and in silico candidate identification.

    a, Crystal structure of human IgCAM3. The colors indicate hydrophobic (blue) or hydrophilic (orange) residues. b, Predicted ΔG scored for the 139,735 molecules docked into the region of small molecule binding. Two groups of molecules with either very low or intermediate ΔG are indicated by symbols that coincide with those in Fig. 2c. The inset shows the IgCAM3 binding pocket (red), with predicted docking of selected small molecules.

  2. Extended Data Fig. 2 Divertin binds to recombinant IgCAM3.

    a, IgCAM3 shown as a ribbon diagram with the location of tryptophan 447 (W447) and three residues in the putative drug-binding pocket, leucine 449 (L449), glutamine 457 (Q457), and aspartic acid 481 (D481). b, Changes in peak fluorescence of wild-type IgCAM3, mutant IgCAM3, and NATA in the presence of increasing concentrations of NSC55937 reveal a shift in the maximum wavelength of wild-type IgCAM3 only. c, Fluorescence emission spectrum of wild-type IgCAM3 in the presence of increasing NSC55937 concentrations demonstrates dose-dependent tryptophan fluorescence quenching and a red shift in the maximum emission wavelength, indicative of NSC55937 binding to IgCAM3. NSC55937 was used at 0 μM (red), 10 μM (orange), 33 μM (yellow), 100 μM (green), 333 μM (blue), and 1000 μM (violet). d, Mutant IgCAM3 (Leu449Arg, Gln457Lys, and Asp481Val) abolishes the ability of NSC55937 to quench IgCAM3 tryptophan fluorescence across all concentrations of divertin. e, Fluorescence emission spectra of the tryptophan analog (1 mM) in the presence of increasing concentrations of NSC55937. No fluorescence quenching occurred. Data are representative of three or more independent experiments with similar results.

  3. Extended Data Fig. 3 Divertin delays the development of experimental inflammatory bowel disease.

    a, Fourteen days after T cell transfer, mice were treated with daily intraperitoneal injections of saline (green) or divertin (red). n = 10 independent animals per condition. The mean ± s.e.m. is shown. **P < 0.01 by unpaired, two-sided t-test with Welch’s correction. b, Divertin-treated mice were protected from the weight loss experienced by saline-treated (green) mice. n = 10 independent animals per condition. the mean ± s.e.m. is shown. **P < 0.01 by unpaired, two-sided t-test with Welch’s correction. c, Divertin significantly increased survival during adoptive transfer colitis. n = 10 independent animals per condition. *P < 0.05 by Gehan–Breslow–Wilcoxon test. d, Intestinal permeability on day 56. Data are normalized to recovery from a healthy wild-type mouse. n = 7 (saline), n = 10 (divertin). **P < 0.01 by unpaired, two-sided t-test with Welch’s correction. e, Mucosal TNF on day 56 was significantly reduced by divertin treatment (red). n = 7 (saline), n = 10 (divertin). *P < 0.05 by unpaired, two-sided t-test with Welch’s correction. f, Colon lengths on day 56. Images of representative colons are shown, with their lengths corresponding to the labels on the y axis. n = 7 (saline) or n = 10 (divertin) independent animals per condition. **P < 0.01 by unpaired, two-sided t-test with Welch’s correction. g, Colonic histopathology scores on day 56. n = 7 (saline) or n = 10 (divertin) independent animals per condition. **P < 0.01 by unpaired, two-sided t-test with Welch’s correction. h, Histopathology shows crypt loss (asterisk) and crypt abscesses (arrow) in the mucosa from a saline-treated mouse and partial goblet cell preservation in the mucosa from a divertin-treated mouse (arrowhead). Bar, 50 μm. The insets show complete cross sections of colon, with the boxes indicating the areas shown at higher magnification. Bar, 250 μm. Images are representative of three independent experiments with similar results. The experiment shown in this figure used female mice as T cell donors and immunodeficient recipients. Results were similar in two independent studies that, in combination, included nine saline-treated and nine divertin-treated male mice.

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