Intestinal barrier function is compromised in enteric and systemic diseases, including infectious enterocolitis, food allergy, celiac disease, graft versus host disease (GvHD), and inflammatory bowel disease (IBD). The co-founders of Thelium Therapeutics discovered the central role of myosin light chain kinase (MLCK) in barrier regulation and demonstrated that targeted intestinal epithelial MLCK inhibition limits experimental IBD and GvHD. Unfortunately, severe toxicities associated with barrier-independent MLCK functions in epithelia and other tissues, e.g., smooth muscle, preclude therapeutic targeting of MLCK enzymatic activity. We recently reported (Graham et al., Nature Medicine, 2019) that a specific MLCK splice variant, MLCK1, is central to barrier regulation and depends on interactions mediated by immunoglobulin-cell adhesion molecule domain 3 (IgCAM3). We solved the IgCAM3 crystal structure, identified a drug binding pocket unique to IgCAM3, and screened a library of ~140,000 drug-like molecules. One compound, Divertin, bound IgCAM3, prevented cytokine-induced MLCK1 recruitment, myosin II regulatory light chain phosphorylation, and barrier dysfunction. Critically, Divertin did not inhibit MLCK enzymatic function, epithelial wound healing, or smooth muscle contraction, and in vivo toxicity studies failed to identify adverse effects. Divertin prevented acute TNF-induced barrier loss in vivo (mice) and ex vivo (human intestinal biopsies) and restored immune-mediated barrier loss in vivo (IL-10 knockout mice). Finally, Divertin delayed onset and prevented progression of experimental immune-mediated (T cell transfer) IBD, as indicated by barrier preservation and restoration, reduced mucosal immune activation, and enhanced survival. Although useful as a tool compound, Divertin lacks characteristics required for a lead compound. This proposal will overcome that obstacle to clinical application of Divertin by discovering lead compounds with similar activities. Cutting edge, physics-based rational design and molecular dynamic simulation methods has already been used to probe an expansive compound library and identify those with predicted high-affinity binding to the MLCK1 IgCAM3 crystal structure. Those molecules will be subjected to a rank order screening funnel to identify compounds with suitable MLCK1 binding affinities, efficacy in preserving and restoring epithelial barrier function, and absence of enzymatic inhibitory activity. These lead compounds will be suitable for optimization and development to facilitate IND-enabling studies for a first-in-class barrier-restorative therapy to manage gastrointestinal and systemic diseases.
Public Health Relevance Statement: PROJECT NARRATIVE The epithelial cells that line the inside of the intestines form a barrier. Although this barrier must be selectively-permeable in order to allow absorption of nutrients and water as well as secretion of waste products, excessive permeability has been implicated as an early step in development of intestinal and systemic diseases. The proposed studies will advance lead discovery efforts towards a first-in-class therapeutic that, by repairing the intestinal barrier, will serve as both a preventative agent and a treatment for active disease.
Project Terms: active method; Actomyosin; Acute; Acute Disease; Adverse effects; Affect; Affinity; Anti-Tumor Necrosis Factor Therapy; base; Binding; Biology; Biopsy; Cardiac Muscle Contraction; Celiac Disease; Cell Adhesion Molecules; Cell Line; Cellular Assay; Characteristics; Chemicals; Chronic; Chronic Disease; clinical application; clinical candidate; Clinical Drug Development; Clinical Trials; Crystallization; cytokine; Data; design; Development; Diarrhea; Disease; Disease model; Docking; Endocytosis; Endothelium; Enteral; Enterocolitis; Enzymes; Epithelial; Epithelial Cells; Epithelium; Event; Experimental Animal Model; Experimental Models; Food Hypersensitivity; Foundations; Functional disorder; Gastrointestinal Diseases; graft vs host disease; Health; Homeostasis; Human; Hydrophobicity; Hypotension; Immune; immune activation; Immunoglobulins; immunomodulatory therapies; Immunosuppressive Agents; improved; In Vitro; in vivo; infection risk; Inflammation; Inflammatory; Inflammatory Bowel Diseases; insight; Interleukin-10; Intestinal Diseases; intestinal epithelium; Intestines; Knockout Mice; Lead; lead candidate; lead optimization; Libraries; Light; Measures; Mediating; Medicine; Methods; Molecular; Mucous Membrane; Mus; Muscle; Muscle Contraction; Myosin Alkali Light Chains; Myosin Light Chain Kinase; Myosin Type II; Nature; non-muscle myosin; novel strategies; novel therapeutics; Nutrient; nutrient absorption; occludin; Outcome; pathogen; Permeability; Pharmaceutical Preparations; Phase; phase 1 study; Phosphorylation; Phosphotransferases; Physics; Physiological; Positioning Attribute; pre-clinical; preservation; prevent; Property; Publishing; recruit; Regulation; repaired; Reporting; restoration; Rho-associated kinase; RNA Splicing; Role; screening; Signal Transduction; simulation; Site; Skeletal Muscle; Small Business Innovation Research Grant; small molecule; Smooth Muscle; Specificity; standard of care; Stimulus; Structure; Systemic disease; systemic toxicity; T-Lymphocyte; Tertiary Protein Structure; Therapeutic; Therapeutic Agents; therapeutic target; Tight Junctions; Tissues; TNF gene; tool; Toxic effect; Variant; Waste Products; Water; Work; Wound Healing
