Identifying novel anti-infectives by high through-put screening in whole animals

描述(申请人提供)：不断涌现的新的和难以治疗的微生物，以及日益增长的多重耐药感染对人类健康构成了严峻的挑战。迫切需要创新的方法来加快发现新的抗感染药物。我们的目标是通过寻找下一代抗感染药物，通过阻断病原体对宿主生理的适应来预防疾病，从而实现抗微生物药物发现的范式转变。为此，我们建议使用整个活体动物对小分子进行高通量筛选。我们已经开发出线虫秀丽线虫的感染模型，可以用来识别治愈其他致命感染的药物。在384孔板中高通量筛选线虫，然后在进化程度更高的模式宿主果蝇黑腹果蝇中进行二次筛选，这增加了分离出对人类有效的药物的可能性。我们的方法适用于许多不同类别的微生物，包括细菌、病毒、真菌和寄生虫。与传统的药物发现相比，它有几个优点：(I)除了识别传统的抗生素外，它还将发现仅在体内显示活性的全新类别的抗感染药物。例如“毒性阻滞剂”和“免疫逃逸阻滞剂”。(Ii)我们的方法是不偏不倚的，不需要事先了解潜在的毒品目标或途径。(Iii)它绕过了目前毒性/药效测试的瓶颈，自动消除有毒化合物(因为它们会杀死线虫)，产生具有体内活性的质量。(Iv)它将确定可防止或减轻微生物耐药性发展的化合物，或可与抗生素治疗相结合，从而提高抗生素疗效。我们预测，我们的方法可以识别抑制毒力的各个方面的化合物：(I)黏附和定植，(Ii)上皮屏障破坏，(Iii)深层组织入侵，(Iv)生物膜形成，(V)避免免疫识别，以及(Vi)调节免疫信号。这些过程背后的一些分子机制在细菌物种中是保守的。为了建立原则证据，我们寻求资金来发现针对铜绿假单胞菌的新的抗感染药物，铜绿假单胞菌是最近出现的几种革兰氏阴性细菌之一，它以一种多重耐药的形式出现，有效的抗生素要么有限，要么得不到。我们计划筛选大量的化合物(25万)，以最大限度地发现新的抗感染药物类别。有希望的化合物将进行表征，在其他革兰氏阴性细菌(克雷伯氏菌、不动杆菌、肠杆菌)中进行有效性测试，并在小鼠感染模型中进行测试。对于前景看好的候选人，我们将尝试分子靶标识别。 与公共卫生相关：导致疾病的微生物对抗生素产生抗药性的速度快于我们发现新的抗生素的速度，这使得许多常见的感染无法治疗，并危及生命。我们项目的目标是找到一种方法来识别新一代抗生素。这些新的复杂药物将通过干扰微生物与人体相互作用的能力来预防疾病，而不是简单地防止细菌生长。

DESCRIPTION (provided by applicant): Continuously emerging new and hard-to-treat microbes, and the growing incidence of multi-drug resistant infections pose formidable challenges to human health. Innovative approaches are urgently needed to speed up the discovery of new anti-infectives. Our aim is to achieve a paradigm shift in antimicrobial drug discovery by finding next generation anti-infectives that prevent disease by blocking pathogen adaptation to host physiology. To this end we propose using whole live animals for high throughput screening of small molecules. We have developed infection models in the nematode Caenorhabditis elegans that can be used to identify drugs that cure otherwise lethal infections. High throughput screening of nematodes in 384-well plates is followed by secondary screening in a more highly evolved model host, the fruit fly Drosophila melanogaster, increasing the likelihood of isolating drugs that will work in humans. Our approach is applicable to many different classes of microorganisms, including bacteria, viruses, fungi and parasites. It has several advantages over traditional drug discovery: (i) In addition to identifying conventional antibiotics, it will uncover entirely new classes of anti-infectives that only exhibit in vivo activity. Examples are "virulence blockers" and "immune escape blockers". (ii) Our approach is unbiased and requires no prior knowledge of potential drug targets or pathways. (iii) It bypasses the current bottleneck of toxicity/efficacy testing by automatically eliminating toxic compounds (because they would kill the nematodes), yielding quality hits with in vivo activity. (iv) It will identify compounds that prevent or mitigate microbial resistance development, or can be combined with antibiotic therapy, thereby increasing antibiotic efficacy. We predict that our approach can identify compounds that inhibit diverse aspects of virulence: (i) adhesion and colonization, (ii) epithelial barrier disruption, (iii) deep tissue invasion, (iv) biofilm formation, (v) avoidance of immune recognition, and (vi) modulation of immune signaling. Some of the molecular mechanisms underlying these processes are conserved across bacterial species. To establish proof-of-principle, we seek funding for discovering new anti-infectives against Pseudomonas aeruginosa, one of several gram-negative bacteria that have recently emerged in a multi-drug resistant form for which efficient antibiotics are either limited or not available. We plan to screen a large number of chemical compounds (250,000) to maximize the discovery of new classes of anti-infectives. Promising compounds will undergo characterization, efficacy testing in other gram-negative bacteria (Klebsiella, Acinetobacter, Enterobacter) and testing in mouse models of infection. For highly promising candidates we will attempt molecular target identification. PUBLIC HEALTH RELEVANCE: Microbes that cause disease are becoming resistant to antibiotics faster than we can find new ones, making many common infections untreatable and life threatening. The goal of our project is to find a way to identify a new generation of antibiotics. Rather than simply preventing bacteria from growing, these new sophisticated drugs will prevent disease by interfering with a microbe's ability to interact with the human body.