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

描述(由申请人提供)：我们的病原体迅速获得耐药性，超过了抗生素发现的缓慢步伐。对于结核分枝杆菌来说，这种需求尤其迫切，那里对大多数标准疗法具有耐药性的广泛耐药结核菌株正在迅速传播。在这个项目中，我们将结合两项创新来开发一个药物发现平台，以确定将随后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；以及高通量筛选特定的抗结核分枝杆菌化合物。今天使用的大多数抗生素都是天然产品或其衍生物，从筛选土壤微生物中获得。从可培养微生物中发现的主要实际问题是已知化合物的巨大背景。与此同时，绝大多数细菌，即99%的物种，不容易在体外生长，因此被称为未培养细菌。我们小组开发了一种通用的方法，通过原位培养来培养未培养的细菌。环境样品，如土壤，与琼脂混合，夹在扩散室的两个半透膜之间，扩散室返回环境。不同生物体的孤立菌落在培养箱中生长，随后重新定位到新的培养箱中，产生了能够在常规培养皿上生长的“驯化”变种。我们的初步发现表明，这是一种很好的新型抗菌剂来源。然而，即使有了这种以前无法获得的资源，大部分的化学努力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种特定物种的化合物，这个问题就可以解决。在结核分枝杆菌的案例中，已经发现了几种合成的化合物-异烟肼、乙硫酰胺和吡津酰胺--对这种微生物具有特殊的作用。到目前为止，还没有描述过专门作用于抗结核分枝杆菌的天然化合物。这意味着一种针对特定抗M的筛选。结核化合物将产生极有可能成为新物质的热门药物。然后，一个特定的屏幕将在很大程度上取代繁琐的去重复。其基本原理是同时对结核分枝杆菌和另一种微生物金黄色葡萄球菌进行筛查。中试筛选显示，从未培养的物种中提取的抗结核分枝杆菌的提取物具有1.5%的极好的特异性命中率。这个屏幕将在拟议的项目中进行优化。为了正确地验证这一筛选，我们需要证明它确实能够识别出专门针对结核分枝杆菌的新化合物。因此，我们将取消打击的复杂性，未知的结构将被确定，它们的行动模式将被建立。一旦在该项目中开发和验证，该屏幕将以HTS格式用于大规模药物发现。一种独特的、尚未开发的来源--未培养的细菌--与特定筛查的结合，可能会导致产生新的化合物来对抗耐药结核分枝杆菌。 公共卫生相关性：在这个项目中，我们将建立一种方法，允许快速识别用于开发治疗结核病的药物的抗微生物化合物。这种方法是基于使用一种独特的抗菌化合物来源--通常不会在实验室生长的细菌，被称为“不可培养的”细菌。我们还将寻找专门针对结核分枝杆菌病原体的化合物，这将避免杀死我们肠道菌群中的有益细菌。

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.