AN HTS ASSAY FOR INHIBITORS OF C. DIFFICLE TOXINS

描述（由申请方提供）：艰难梭菌相关疾病（CDAD）是一种以肠道损伤和显著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，特别是在社区中其他健康的个人中。在发病率增加的同时，这种疾病也变得更加严重，发病率和死亡率都有记录地增加。目前CDAD的抗生素治疗通常无效，导致寻找替代治疗方法。主要毒力因子的研究。艰难梭菌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是C.艰难的毒力这两种酶都将葡萄糖从UDP-葡萄糖（UDPG）转移到宿主GT3蛋白，导致其失活和随后的细胞死亡。这两种酶还具有将UDPG切割成游离UDP和葡萄糖的糖水解酶活性。 目前还没有专门针对这些毒素的治疗方法。由于C.艰难梭菌主要是由于毒素B，我们的目标是确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，并将这两种方法应用于384孔板。在进行高通量筛选之前，将在中试HTS中对这些测定进行比较。将优化确证性和二次试验，以评价HTS鉴别的TcdB抑制剂。在该提案的第二年，我们计划将我们的检测方法加入分子库筛选探针网络（MLSPN）。长期目标是在体外和体内C.艰难梭菌相关疾病。我们预计，其中最有希望的将被开发为针对CDAD的潜在疗法。

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.