AN HTS ASSAY FOR INHIBITORS OF C. DIFFICLE TOXINS

描述（由申请人提供）：艰难梭菌相关疾病（CDAD）是一种以肠道损伤和明显炎症反应为特征的疾病。直到最近，这种疾病通常见于住院患者或长期服用抗生素的患者。然而，这种疾病正变得越来越普遍，特别是在社区中健康的个人中。随着患病率的增加，该病变得越来越严重，有充分证据表明发病率和死亡率都在增加。目前的抗生素治疗CDAD往往是无效的，导致寻找替代治疗方法。艰难梭菌的主要毒力因子是一对密切相关的毒素a和B（也称为TcdA和TcdB）。最近的体内研究表明，TcdB对艰难梭菌的毒力至关重要。这两种酶将葡萄糖从udp -葡萄糖（UDPG）转移到宿主GTPase蛋白，导致其失活和随后的细胞死亡。这两种酶还具有糖水解酶活性，可以裂解UDPG以释放UDP和葡萄糖。目前还没有专门针对这些毒素的疗法。由于艰难梭菌引起的肠道损伤和临床疾病主要是由于毒素B，我们的目标是鉴定这种毒素的特异性抑制剂。我们克隆并表达了TcdB的催化结构域，开发了两种检测其活性的方法，并将这两种方法都适用于384孔格式。在进行高通量筛选之前，这些检测将在试点HTS中进行比较。将优化验证和二级分析，以评估HTS鉴定的TcdB抑制剂。在该提案的第二年，我们计划将我们的分析进入分子文库筛选探针网络（MLSPN）。长期目标是在结构上优化艰难梭菌相关疾病的体外和体内模型中的毒素抑制剂。我们预计其中最有希望的将被开发为潜在的治疗CDAD的方法。

DESCRIPTION (provided by applicant): Clostridium difficile-associated disease (CDAD) is an illness characterized by damage to the intestine and a marked inflammatory response. Until recently the disease was generally seen in hospitalized patients or those on antibiotics for prolonged periods. However, the disease is becoming increasingly prevalent, particularly among otherwise healthy individuals in the community. Coincident with its increased prevalence, the disease is becoming more severe with well documented increases in morbidity and mortality. Current antibiotic treatment of CDAD is often ineffective, leading to the search for alternate therapeutic approaches. The cardinal virulence factors of C. difficile are a pair of closely related toxins A and B (also known as TcdA and TcdB). Recent in vivo studies demonstrate TcdB to be the most essential to C. difficile virulence. Both enzymes transfer glucose from UDP-glucose (UDPG) to host GTPase proteins, resulting in their inactivation and subsequent cell death. Both enzymes also possess a glycohydrolase activity that cleaves UDPG to free UDP and glucose. There are currently no therapies that specifically target these toxins. Since the intestinal damage and clinical disease caused by C. difficile is predominantly due to toxin B, our goal is to identify specific inhibitors of this toxin. We have cloned and expressed the catalytic domain of TcdB, developed two assays to detect its activity, and have adapted both assays to 384-well format. Before proceeding with high throughput screening, these assays will be compared in a pilot HTS. Confirmatory and secondary assays will be optimized for the evaluation of TcdB inhibitors identified by HTS. In the second year of this proposal we plan to enter our assay into the Molecular Libraries Screening Probes Network (MLSPN). Longer term goals are to structurally optimize toxin inhibitors in vitro and in vivo models of C. difficile associate disease. We anticipate that the most promising of these will be developed as potential therapies against CDAD.