A synergy-based therapy against C. difficile

描述(申请人提供)：肠道厌氧菌艰难梭菌感染的发病率增加，加上耐药高毒力菌株的传播，要求开发这种潜在致命病原体的新疗法。该项目的目标是开发一种新的治疗艰难梭菌的方法，其基础是耐受性良好的抗菌剂小连素和一种多药外排泵(MDR)抑制剂的组合。黄连素是加拿大水华的主要成分，我们以前的研究确定了它对细菌病原体的疗效受到MF家族多药耐药性的限制。然而，多药耐药抑制剂使黄连素对包括艰难梭菌在内的革兰氏阳性细菌的活性增加了60倍以上。黄连素和多药耐药抑制剂的结合物也得到了类似的结果。由于黄连素唯一已知的耐药机制是多药耐药外排，它与多药耐药泵抑制剂的结合，无论是单独的还是作为杂化分子，都可以产生一种强大的抗菌剂。重要的是，黄连素/多药耐药抑制剂组合对艰难梭菌的静止细胞具有很强的杀菌活性，而常用的甲硝唑和万古霉素只对生长中的细菌有活性。艰难梭菌在达到静止状态时会产生孢子，这是导致感染复发的原因。通过杀死静止细胞，黄连素/多药耐药抑制剂组合防止孢子形成。这表明，通过防止复发，联合抗菌药物可能比现有的治疗方法具有关键优势。在化合物库中发现了抗MF MDRs的抑制剂的可能性很高(~5%)，这为挑选具有吸引人的性质(包括吸收性能较差)的先导化合物提供了独特的机会。发现多药耐药抑制剂的可能性非常高，这为合理管理耐药性提供了独特的机会。由于耐药性的发展，传统的抗菌药最终会失效。我们方法的优势是，从大量的热门化合物中可以相对容易地开发出新类别的MDR抑制剂。通过这种方式，我们将能够保持在病原体抗性方面的领先地位。虽然发现MDR抑制剂HITS的可能性很高，但将它们开发成先导化合物也是一个挑战，因为在体内测试大量化合物的毒性和有效性是不切实际的。我们最近描述了一种线虫的全动物筛选，它有助于克服这些发展瓶颈。我们将使用这个模型来快速识别有吸引力的线索，这些线索将为我们的药物开发流水线提供支持。铅，包括我们已经确定的那些，将在体外(效力、光谱、细胞毒性、吸光度)进行评估，然后在小鼠身上进行毒性评估，并在艰难梭菌感染的仓鼠模型中进行疗效评估。这个多学科项目是抗菌化学、药物化学、艰难梭菌生物学、宿主-病原体相互作用和兽医专家之间的合作，他们在该项目的初步阶段进行了合作。

DESCRIPTION (provided by applicant): The increasing incidence of infections by the intestinal anaerobe Clostridium difficile coupled with the spread of drug resistant highly virulent strains dictates the need for developing new therapies for this potentially lethal pathogen. The goal of this project is to develop a novel therapy against C. difficile based on a combination of the well-tolerated antimicrobial berberine and an inhibitor of Multidrug Efflux Pumps (MDRs). Berberine is the principal component of Hydrastis canadensis (Goldenseal), and our previous research determined that its efficacy against bacterial pathogens is limited by MDRs of the MF family. However, MDR inhibitors increase the activity of berberine against gram positive bacteria, including C. difficile, by more than 60 fold. Similar results were found for a conjugate of berberine and an MDR inhibitor. Since the only known mechanism of resistance to berberine is MDR efflux, its combination with an MDR pump inhibitor, either separately or as a hybrid molecule, can result in a powerful antimicrobial. Importantly, the berberine/MDR inhibitor combination had strong bactericidal activity against stationary cells of C. difficile, in contrast to commonly used metronidazole and vancomycin, which were only active against growing bacteria. Upon reaching stationary state, C. difficile produces spores, which are responsible for the relapse of the infection. By killing stationary cells, berberine/MDR inhibitor combination prevents spore formation. This suggests that the combination antimicrobial may have a critical advantage over existing therapeutics by preventing relapse. Inhibitors against MF MDRs are found with high probability in compound libraries (~5%), presenting a unique opportunity to pick and choose leads with attractive properties (including poor absorption). The very high probability of finding MDR inhibitors provides a unique opportunity to rationally manage drug resistance. Conventional antimicrobials eventually fail due to resistance development. The advantage of our approach is that new classes of MDR inhibitors can be relatively easily developed from an enormous base of hit compounds. In this manner, we will be able to stay ahead of pathogen resistance. While finding MDR inhibitor hits has a high probability, developing them into leads presents a challenge as well, since it has been impractical to test a large number of compounds for toxicity and efficacy in vivo. We have recently described a whole-animal screen in C. elegans, that helps overcome these bottlenecks in development. We will use this model to rapidly identify attractive leads which will feed our drug development pipeline. Leads, including those we already identified, will be evaluated in vitro (potency, spectrum, cytotoxicity, absorbance) and then in mice for toxicity, and in a hamster model of C. difficile infection for efficacy. This multidisciplinary project is a collaboration between experts in antimicrobial chemotherapy, medicinal chemistry, C. difficile biology, host-pathogen interactions and veterinary medicine who have worked together on preliminary stages of this project.