A High-Throughput Screen for Specific Anti-M. tuberculosis Compounds

描述（由申请人提供）：抗生素发现的缓慢步伐正在被我们的病原体快速获得耐药性所超越。在M的情况下，这种需要尤其迫切。对大多数标准疗法具有抗药性的广泛耐药结核菌株正在迅速蔓延。在这个项目中，我们将联合收割机结合两项创新来开发一个药物发现平台，用于识别随后将被开发成治疗结核病的疗法的线索：使用未培养的细菌作为新型抗菌剂的独特来源;以及高通量筛选特异性抗结核化合物。 目前使用的大多数抗生素都是通过筛选土壤微生物获得的天然产物或其衍生物。从可培养的微生物中发现的主要实际问题是已知化合物的巨大背景。同时，绝大多数细菌，99%的物种，不容易在体外生长，被称为未培养的。我们的小组开发了一种通过原位培养来培养未培养细菌的通用方法。将环境样品如土壤与琼脂混合，并夹在扩散室的两个半透膜之间，将其返回到环境中。不同生物体的隔离菌落在腔室中生长，随后重新接种到新的腔室中产生能够在体外常规培养皿上生长的“驯化”变体。我们的初步研究结果表明，这是一个很好的来源，新的抗菌剂。然而，即使有了这种以前无法获得的资源，大部分化学工作仍然浪费在重新发现已知化合物上。我们的理由是，如果发现的重点是一个物种特异性化合物，这个问题可以得到解决。如果M。在结核病中，已经发现几种合成化合物特异性地作用于该生物体- INH、乙硫异烟胺和吡嗪酰胺。天然化合物特异性作用于M。迄今为止，结核病尚未被描述。这意味着，屏幕上的具体抗M。结核病化合物将产生具有成为新物质的高概率的命中物。一个特定的屏幕将在很大程度上取代费力的去复制。基本原理是与M平行筛选。结核和另一种微生物S.金黄色。中试筛选表明，未培养种的提取物对M.结核该屏幕将在拟议项目中进行优化。为了正确地验证筛选，我们需要证明它确实能够识别特异性对抗M的新化合物。结核因此，我们将去复制命中，未知的结构将被确定，他们的行动模式将被建立。一旦在该项目中开发和验证，该筛选将以HTS格式用于大规模药物发现。一个独特的，未开发的来源-未培养的细菌-和一个特定的屏幕相结合，可能会导致新的化合物，以打击抗药性M。结核 公共卫生相关性：在这个项目中，我们将建立一种方法，可以快速识别抗菌化合物，用于开发治疗结核病的药物。该方法基于使用独特的抗菌化合物来源--通常不会在实验室中生长且被称为“不可培养”的细菌。我们还将寻找对病原体M有特异性作用的化合物。肺结核，这将避免杀死我们肠道植物群中的有益细菌。

DESCRIPTION (provided by applicant): The slow pace of antibiotic discovery is being outmatched by rapid acquisition of resistance by our pathogens. The need is especially acute in the case of M. tuberculosis, where strains of XDR-TB resistant to the majority of standard therapeutics are rapidly spreading. In this project, we will combine two innovations to develop a drug discovery platform for identifying leads that will be subsequently developed into therapeutics for treating tuberculosis: the use of uncultured bacteria as a unique source of novel antimicrobials; and a high-throughput screen for specific anti-Mtb compounds. Most antibiotics in use today are natural products or their derivatives, obtained from screening soil microorganisms. The main practical problem with discovery from culturable microorganisms is the enormous background of known compounds. At the same time, the vast majority of bacteria, 99% of species, do not readily grow in vitro and are known as uncultured. Our group developed a general method to grow uncultured bacteria by cultivating them in situ. An environmental sample such as soil is mixed with agar and sandwiched between two semi-permeable membranes of a diffusion chamber which is returned to the environment. Isolated colonies of diverse organisms grow in the chamber, and subsequent reinoculation to new chambers produces "domesticated" variants capable of growing on regular Petri dishes in vitro. Our preliminary findings show that this is an excellent source of novel antimicrobials. However, even with this previously inaccessible resource most of the chemistry effort is still wasted on rediscovery of known compounds. We reason that the problem can be resolved if discovery is focused on a species-specific compound. In case of M. tuberculosis, several synthetic compounds have been discovered that specifically act against this organism - INH, ethionamide and pyrazinamide. Natural compounds specifically acting against M. tuberculosis have not been described so far. This means that a screen for specific anti-M. tuberculosis compounds will produce hits that will have a high probability of being novel substances. A specific screen will then largely replace the laborious dereplication. The rationale is to screen in parallel against M. tuberculosis and a different organism, S. aureus. A pilot screen showed an excellent specific hit rate of 1.5% for extracts from uncultured species acting against M. tuberculosis. This screen will be optimized in the proposed project. In order to properly validate the screen, we will need to demonstrate that it is indeed capable of identifying novel compounds acting specifically against M. tuberculosis. We will therefore dereplicate the hits, the structure of unknowns will be determined, and their mode of action will be established. Once developed and validated in this project, the screen will be used in an HTS format for large-scale drug discovery. A combination of a unique, untapped source - uncultured bacteria - and a specific screen is likely to lead to novel compounds to combat drug-resistant M. tuberculosis. PUBLIC HEALTH RELEVANCE: In this project, we will establish a method that allows to rapidly identify antimicrobial compounds for developing drugs to treat tuberculosis. The method is based on using a unique source of antimicrobial compounds - bacteria that do not normally grow in the lab and are known as "unculturable". We will also look for compounds that act specifically against the pathogen M. tuberculosis, which will avoid killing of the good bacteria of our gut flora.