Development of non-antibiotic therapeutics for Clostridium difficile infection (CDI)

项目摘要 C.艰难梭菌感染（CDI）是由使用广谱抗生素引起的， 肠道的植物群，并允许革兰氏阳性厌氧病原体茁壮成长。发病率上升， 疾病的严重性需要新的最佳治疗策略。此外，答复率下降 甲硝唑，高复发率与使用多种抗生素，并出现多种抗生素 耐药细菌的出现表明迫切需要开发新的治疗方法。一个范式的转变， 感染性疾病的治疗，包括CDI，是必要的，以防止抗生素耐药性的增加， 应考虑抗生素的替代品。 该项目的目标是开发一种新型的非抗生素 抗C.艰难梭菌的基础上抑制的主要细菌毒力因子介导的 疾病这些毒素通过变构激活在肠上皮细胞内被激活 它们的半胱氨酸蛋白酶结构域（CPD）的真核生物特异性小分子肌醇六磷酸 （InsP6）。我们已经开发了一种基于活性的探针，可以报告毒素CPD激活。利用这个探测器， 我们进行了高通量荧光偏振筛选，以确定新的小分子抑制剂， 毒素筛选显示了大量新的先导化合物以及现有的第二阶段 具有纳摩尔活性的临床药物。我们已经用临床药物来证明 在小鼠中使用致炎和感染模型阻断毒素功能的治疗价值。这些研究 已经证实CPD的抑制阻断了宿主组织中病原体介导的毒性。然而，其穷人 药理学性质加上其广泛的靶点选择性使其对于临床应用并不理想。 发展CDI。因此，本项目将在项目的前3年重点确定可行的铅 这些化合物具有改进的效力、选择性、溶解性和细胞摄取。我们将专注于临床领先 化合物以及几个新的化学实体（NCE）确定在我们的HTS努力。我们将确定三个 化学上区分铅分子，然后在小鼠和仓鼠CDI模型中进行验证。我们将 将其中一种分子（保留其他分子作为备份）推进到配方研究中，最终目标是 确定一个单一的先导分子和配方策略，可以进入IND使能研究， 完成这个项目。

Project Summary C. difficile infection (CDI) is triggered by use of broad-spectrum antibiotics which disrupts the natural microbial flora of the gut and allows the Gram-positive anaerobic pathogen to thrive. The increased incidence and severity of the disease requires new strategies for optimal treatment. Furthermore, decreased response rates to metronidazole, high recurrence rates with the use multiple antiboitics, and emergence of multiple antibiotic resistant bacteria demonstrate the urgent need to develop new therapies. A paradigm shift in the general treatment of infectious diseases, including CDI, is necessary to prevent an increase in antibiotic resistance and alternatives to antibiotics should be considered.  The goal of this project is to develop a novel non-antibiotic therapy against C. difficile based on the inhibition of the major bacterial virulence factors that mediate the disease. These toxins are post-translationally activated inside intestinal epithelial cells via allosteric activation of their cysteine protease domain (CPD) by the eukaryote-specific small molecule inositol-hexakisphosphate (InsP6). We have developed an activity-based probe that can report on toxin CPD activation. Using this probe, we conducted a high-throughput fluorescence polarization screen to identify novel small-molecule inhibitors of the toxins. The screen revealed a significant number of novel lead compounds as well as an existing phase II clinical drug with nanomolar activity against the toxin. We have used the clinical drug to demonstrate the therapeutic value of blocking toxin function, using both toxigenic and infection models in mice. These studies have confirmed that inhibition of the CPD blocks pathogen-mediated toxicity in host tissues. However, its poor pharmacological properties coupled with its broad target selectivity makes it less than ideal for clinical development for CDI. Therefore, this project will focus in the first 3 years of the project to identify viable lead compounds with improved potency, selectivity, solubility and cellular uptake. We will focus on the clinical lead compound as well as several novel chemical entities (NCEs) identified in our HTS efforts. We will identify three chemically distinct lead molecules and then perform validation in mouse and hamster models of CDI. We will advance one of these molecules (keeping the others as backups) into formulation studies, with the end goal being to identify a single lead molecule and formulation strategy that can move into IND enabling studies after completion of this project.