BACKGROUND & AIMS Mutations in the tetratricopeptide repeat domain 7A gene (TTC7A) cause intestinal epithelial and immune defects. Patients can become immune deficient and develop apoptotic enterocolitis, multiple intestinal atresia, and recurrent intestinal stenosis. The intestinal disease in patients with TTC7A deficiency is severe and untreatable, and it recurs despite resection or allogeneic hematopoietic stem cell transplant. We screened drugs for those that prevent apoptosis of in cells with TTC7A deficiency and tested their effects in an animal model of the disease. METHODS We developed a high-throughput screen to identify compounds approved by the US Food and Drug Administration that reduce activity of caspases 3 and 7 in TTC7A-knockout (TTC7A-KO) HAP1 (human haploid) cells and reduce the susceptibility to apoptosis. We validated the effects of identified agents in HeLa cells that stably express TTC7A with point mutations found in patients. Signaling pathways in cells were analyzed by immunoblots. We tested the effects of identified agents in zebrafish with disruption of ttc7a, which develop intestinal defects, and colonoids derived from biopsy samples of patients with and without mutations in TTC7A. We performed real-time imaging of intestinal peristalsis in zebrafish and histologic analyses of intestinal tissues from patients and zebrafish. Colonoids were analyzed by immunofluorescence and for ion transport. RESULTS TTC7A-KO HAP1 cells have abnormal morphology and undergo apoptosis, due to increased levels of active caspases 3 and 7. We identified drugs that increased cell viability; leflunomide (used to treat patients with inflammatory conditions) reduced caspase 3 and 7 activity in cells by 96%. TTC7A-KO cells contained cleaved caspase 3 and had reduced levels of phosphorylated AKT and X-linked inhibitor of apoptosis (XIAP); incubation of these cells with leflunomide increased levels of phosphorylated AKT and XIAP and reduced levels of cleaved caspase 3. Administration of leflunomide to ttc7a-/- zebrafish increased gut motility, reduced intestinal tract narrowing, increased intestinal cell survival, increased sizes of intestinal luminal spaces, and restored villi and goblet cell morphology. Exposure of patient-derived colonoids to leflunomide increased cell survival, polarity, and transport function. CONCLUSIONS In a drug screen, we identified leflunomide as an agent that reduces apoptosis and activates AKT signaling in TTC7A-KO cells. In zebrafish with disruption of ttc7a, leflunomide restores gut motility, reduces intestinal tract narrowing, and increases intestinal cell survival. This drug might be repurposed for treatment of TTC7A deficiency.

中文翻译：

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药物筛选可鉴定来氟米特用于治疗TTC7A缺乏引起的炎症性肠病。

背景与目的四三肽重复结构域7A基因（TTC7A）中的突变引起肠道上皮和免疫缺陷。患者可能会出现免疫缺陷，并发生凋亡性小肠结肠炎，多发性肠闭锁和复发性肠狭窄。TTC7A缺乏症患者的肠道疾病严重且无法治愈，尽管切除或同种异体造血干细胞移植后仍会复发。我们筛选了可预防TTC7A缺乏的细胞凋亡的药物，并在该疾病的动物模型中测试了它们的作用。方法我们开发了一种高通量筛选技术，以鉴定获得美国食品和药物管理局批准的化合物，这些化合物可降低TTC7A敲除（TTC7A-KO）HAP1（人类单倍体）细胞中胱天冬氨酸蛋白酶3和7的活性，并降低对细胞凋亡的敏感性。我们验证了稳定表达患者中点突变的TTC7A的HeLa细胞中已鉴定药物的作用。通过免疫印迹分析细胞中的信号传导途径。我们测试了斑马鱼中鉴定出的药物对ttc7a的破坏（这会导致肠道缺陷）以及源自TTC7A有无突变患者活检样本的类结肠的作用。我们对斑马鱼的肠蠕动进行了实时成像，并对患者和斑马鱼的肠组织进行了组织学分析。通过免疫荧光和离子转运分析结肠。结果TTC7A-KO HAP1细胞具有异常的形态并发生凋亡，这是由于活性胱天蛋白酶3和7水平升高所致。来氟米特（用于治疗患有炎症的患者）使细胞中的半胱天冬酶3和7活性降低了96％。TTC7A-KO细胞含有裂解的caspase 3，磷酸化的AKT和X连锁的凋亡抑制剂（XIAP）含量降低。将这些细胞与来氟米特一起孵育会增加磷酸化的AKT和XIAP的水平，并降低裂解的caspase的水平3。将来氟米特用于ttc7a-/-斑马鱼会增加肠蠕动，减少肠道狭窄，增加肠道细胞的存活率，增加肠腔的大小，并恢复绒毛和杯状细胞的形态。患者来源的类固醇暴露于来氟米特可增加细胞存活率，极性和转运功能。结论在药物筛查中，我们鉴定来氟米特为可减少细胞凋亡并激活TTC7A-KO细胞中AKT信号的物质。在具有ttc7a破坏的斑马鱼中，来氟米特可恢复肠蠕动，减少肠道狭窄，并增加肠道细胞存活率。该药物可能被重新用于治疗TTC7A缺乏症。