Inhibitors of Intestinal Epithelial Cell Lysosomal-Mediated Necrosis

肠上皮在屏障功能中起着至关重要的作用。这些细胞经常受到损伤， 在炎症性肠病（IBD）样疾病中进展至死亡。上皮细胞损失通常通过以下途径发生： 细胞死亡表型的混合物，如凋亡或坏死性凋亡，但以溶酶体介导的 (LM)当受到严重的伤害时就会坏死。临床上，LM坏死表现为大片上皮细胞 粘膜脱落进入肠腔。保持正常上皮细胞的活力和功能将有助于 改善IBD样疾病的并发症。最初被认为是偶然的和不可逆转的，我们表明， LM坏死遵循分子程序并且是可逆的。我们假设， 高通量、高内容体内筛选（HTS/HCS）LM坏死小分子解毒剂将引发 临床前药物发现管道的形式肠上皮细胞死亡，目前缺乏任何手段 化学预防。不幸的是，哺乳动物系统的费用和复杂性使它们 不适合HTS/HCS格式。此外，这些系统存在混合的细胞死亡表型，从而 使纯LM坏死抑制剂的分离复杂化。我们发现了一个C。elegans strain null for a inhibitor of 溶酶体半胱氨酸蛋白酶。这些动物对LM肠坏死高度且唯一易感 当受到IBD样疾病中发生的许多有害刺激时。这种细胞死亡途径是 在进化上是保守的，如来自同源半胱氨酸缺失小鼠的胎儿肠细胞外植体培养物， 蛋白酶抑制剂也优先经历LM肠坏死，即使当暴露于凋亡或 坏死性下垂刺激我们的实验室率先使用了突变体C。elegans菌株进行活体动物， HTS/HCS药物发现活动（美国专利US 8809617 B2）。这些筛选的化合物具有 已经在类似的哺乳动物疾病模型中证明了有效性。利用这种强大的技术，我们 建议用我们的变种人C elegans菌株完善HTS/HCS测定LM坏死，并进行 大规模筛选肠上皮LM坏死小分子抑制剂。有希望的热门歌曲已经 在一个小规模的试点筛选中，使用FDA批准的药物库进行了鉴定。命中将使用 涉及线虫、哺乳动物细胞系和小鼠肠上皮细胞的二级试验数量 模型人坏死性小肠结肠炎的损伤方案。进行活体动物LM坏死的能力 基于表型的新疗法筛选对于IBD样疾病的发作是前所未有的， 广泛的上皮细胞损伤。本提案是根据PA-16-374，测定开发和 筛选以发现NIDDK感兴趣疾病的治疗或成像剂。

The intestinal epithelium plays a crucial role in barrier function. These cells are often injured and progresses to death in inflammatory bowel disease (IBD)-like disorders. Epithelial cell loss typically occurs via a mixture of cell death phenotypes such as apoptosis or necroptosis, but is dominated by lysosomal-mediated (LM) necrosis when the insult is severe. Clinically, LM necrosis is manifest by large segments of the epithelial lining sloughing into the intestinal lumen. Preservation of normal epithelial cell viability and function will help ameliorate the complications of IBD-like disorders. Initially perceived as haphazard and irreversible, we show that LM necrosis follows a molecular program and is reversible. We hypothesize that the development of a high-throughput, high-content in vivo screen (HTS/HCS) for small molecule antidotes to LM necrosis will prime the preclinical drug discovery pipeline for a form of intestinal epithelial cell death that currently lacks any means of chemoprophylaxis. Unfortunately, the expense and complexity of mammalian systems make them unsuitable for HTS/HCS formats. Moreover, these systems present with mixed cell death phenotypes, thereby complicating the isolation of pure LM necrosis inhibitors. We identified a C. elegans strain null for an inhibitor of lysosomal cysteine proteases. These animals are highly and exclusively susceptible to LM intestinal necrosis when stressed by many of the noxious stimuli occurring in IBD-like disorders. This cell death pathway is evolutionarily conserved, as fetal intestinal cell explant cultures from mice null for the homologous cysteine protease inhibitor also preferentially undergo LM intestinal necrosis, even when exposed to apoptotic or necroptotic stimuli. Our laboratory pioneered the use of mutant C. elegans strains to conduct live-animal, HTS/HCS drug discovery campaigns (US Patent US8809617 B2). Compounds from these screens have already proved effective in analogous mammalian models of disease. Using this robust technology, we propose to use our mutant C. elegans strain to perfect an HTS/HCS assay for LM necrosis and conduct a large-scale screen for small molecule inhibitors of intestinal epithelial LM necrosis. Promising hits have already been identified in a small pilot screen using a library of FDA-approved drugs. Hits will be validated using a number of secondary assays involving nematodes, mammalian cell lines and a mouse intestinal epithelial injury protocol that models human necrotizing enterocolitis. The ability to conduct a live-animal, LM necrosis phenotype-based screens for new therapeutics is unprecedented for episodes of IBD-like diseases marked by extensive epithelial cell injury. This proposal was prepared in response to PA-16-374, Assay Development and Screening to Discover Therapeutic or Imaging Agents for Diseases of interest to NIDDK.