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

项目摘要 艰难梭菌感染(CDI)是由使用广谱抗生素引发的，这种抗生素破坏了自然微生物。 肠道菌群，使革兰氏阳性厌氧菌茁壮成长。增加的发病率和 这种疾病的严重性需要新的战略来实现最佳治疗。此外，响应率降低 对甲硝唑来说，复发率高，配合使用多种抗菌药物，并出现多种抗生素 耐药细菌表明迫切需要开发新的治疗方法。总体上的范式转变 治疗传染病，包括CDI，是必要的，以防止抗生素耐药性的增加和 应该考虑抗生素的替代品。这个项目的目标是开发一种新型的非抗生素 基于抑制介导艰难梭菌的主要细菌毒力因子的艰难梭菌的治疗 疾病。这些毒素通过变构激活在肠上皮细胞内被翻译后激活 真核生物特异性小分子肌醇-六氢磷酸对其半胱氨酸蛋白酶结构域(CPD)的影响 (InsP6)。我们已经开发了一种基于活性的探针，可以报告毒素CPD的激活。使用这个探测器， 我们进行了高通量的荧光偏振筛选，以确定新的小分子抑制剂 毒素。筛查显示了大量新的先导化合物以及现有的第二相 具有纳摩尔抗毒素活性的临床药物。我们已经用临床药物证明了 用小鼠产毒和感染模型阻断毒素功能的治疗价值。这些研究 已证实抑制CPD可阻断病原体在宿主组织中的毒性。然而，它的贫穷 药理特性加上其广泛的靶点选择性，使其在临床上不太理想 CDI的发展。因此，该项目将重点放在项目的前3年，以确定可行的领导 具有更好的效力、选择性、溶解性和细胞摄取的化合物。我们将专注于临床领先 化合物以及在我们的HTS工作中发现的几种新的化学实体(NCEs)。我们将确定三个 化学上不同的铅分子，然后在CDI的小鼠和仓鼠模型中进行验证。我们会 将这些分子中的一个(将其他分子作为备份)推进到配方研究中，最终目标是 是确定单一的铅分子和配方策略，可以进入IND使能研究之后 完成这一项目。

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.