A synergy-based therapy against C. difficile

描述（由申请人提供）：肠道厌氧菌艰难梭菌感染的发生率不断增加，加上耐药高毒力菌株的传播，表明需要开发针对这种潜在致命病原体的新疗法。该项目的目标是基于耐受性良好的抗菌小檗碱和多药物外排泵（mdr）抑制剂的组合，开发一种针对艰难梭菌的新疗法。小檗碱是加拿大Hydrastis canadensis （Goldenseal）的主要成分，我们之前的研究确定其对细菌病原体的作用受到MF家族mdr的限制。然而，耐多药抑制剂可使小檗碱对革兰氏阳性细菌（包括艰难梭菌）的活性提高60倍以上。小檗碱和耐多药抑制剂的共轭物也发现了类似的结果。由于对小檗碱的耐药性的唯一已知机制是耐多药外排，它与耐多药泵抑制剂的组合，无论是单独的还是作为混合分子，都可以产生强大的抗菌素。重要的是，小檗碱/MDR抑制剂联合对艰难梭菌的静止细胞有很强的杀菌活性，而常用的甲硝唑和万古霉素仅对生长细菌有活性。在达到静止状态后，艰难梭菌产生孢子，这是导致感染复发的原因。通过杀死静止的细胞，小檗碱/MDR抑制剂联合阻止孢子形成。这表明联合抗菌素可能比现有的治疗方法具有预防复发的关键优势。针对MF mdr的抑制剂在化合物文库中被发现的概率很高（~5%），这为挑选具有吸引性质（包括吸收差）的引线提供了独特的机会。发现耐多药抑制剂的高概率为合理管理耐药性提供了独特的机会。常规抗菌剂最终由于耐药性的发展而失效。我们的方法的优点是，新的耐多药抑制剂可以相对容易地从一个巨大的基础击中化合物开发。通过这种方式，我们将能够领先于病原体耐药性。虽然发现MDR抑制剂命中的概率很高，但将它们开发成先导药物也面临着挑战，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。我们最近描述了秀丽隐杆线虫的全动物筛选，这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，这将为我们的药物开发管道提供支持。我们将在体外（效价、光谱、细胞毒性、吸光度）评估导联剂，然后在小鼠身上进行毒性评估，在艰难梭菌感染的仓鼠模型中进行疗效评估。这个多学科项目是由抗菌化疗、药物化学、艰难梭菌生物学、宿主-病原体相互作用和兽医学方面的专家合作完成的，他们在项目的初步阶段就在一起工作。

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.