A Novel Approach to Restore Epithelial Barrier Homeostasis to Treat Inflammatory Bowel Disease

摘要 肠道和全身疾病，包括传染性疾病，肠道屏障功能受损 小肠结肠炎、食物过敏、腹腔疾病、移植物抗宿主病(GvHD)和炎症性疾病 肠道疾病(IBD)。钍治疗公司的联合创始人发现了 肌球蛋白轻链激酶(MLCK)在屏障调节和靶向肠道中的作用 上皮MLCK抑制可限制实验性IBD和GvHD。不幸的是，严重的毒副作用 与上皮和其他组织中的非屏障MLCK功能有关，如Smooth 肌肉，排除MLCK酶活性的治疗靶点。我们最近报道了 (Graham等人，自然医学，2019)认为特定的MLCK剪接变异体MLCK1是 屏障调节及其依赖于免疫球蛋白-细胞黏附的相互作用 分子结构域3(IgCAM3)。我们解析了IgCAM3的晶体结构，鉴定了药物结合 Pocket是IgCAM3独有的，并筛选了一个约140,000个类药物分子的文库。我们的工具 Divertin，结合IgCAM3的化合物，阻止细胞因子诱导的MLCK1募集，肌球蛋白II 调节轻链磷酸化和屏障功能障碍。关键的是，Divertin并没有抑制 MLCK酶功能，上皮伤口愈合，或平滑肌收缩，以及体内 毒性研究未能确定不良反应。Divertin预防急性肿瘤坏死因子诱导的屏障 体内(小鼠)和体外(人类肠道活检)丢失，并恢复免疫介导性 体内屏障丧失(IL-10基因敲除小鼠)。最后，Divertin延迟起效并预防 屏障提示实验性免疫介导(T细胞转移)IBD的研究进展 保存和修复，降低粘膜免疫活性，提高存活率。在一个 第一阶段SBIR，我们通过发现具有改进的Hit化合物来推进该计划 活动。命中化合物是通过严格的等级顺序筛选漏斗发现的 鉴定具有合适的MLCK1结合亲和力、保存和修复效果的化合物 上皮屏障功能，缺乏酶抑制活性。在这份第二阶段提案中， 这些Hit化合物将通过迭代结构活性关系研究和 在IBD小鼠模型上进行毒性和疗效测试。这项工作将促进IND的启用 一流和一流的障碍恢复性疗法的研究 胃肠道和全身疾病。

SUMMARY 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. Our tool 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. In a Phase I SBIR, we advanced this program through the discovery of hit compounds with improved activities. Hit compounds were discovered through a rigorous 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. In this Phase II proposal, these hit compounds will be optimized with iterative structure activity relationship studies and tested for toxicity and efficacy in mouse models of IBD. This work will facilitate IND-enabling studies for a first-in-class and best-in-class barrier-restorative therapy to manage gastrointestinal and systemic diseases.