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被我们的病原体快速获得耐药性所取代。在结核分枝杆菌的情况下，这种需求尤其迫切，因为对大多数标准治疗药物具有耐药性的广泛耐药结核菌株正在迅速蔓延。在这个项目中，我们将结合两项创新来开发一个药物发现平台，以确定随后将开发成治疗结核病的疗法的线索：利用未培养的细菌作为新型抗菌剂的独特来源；以及特异性抗结核分枝杆菌化合物的高通量筛选。目前使用的大多数抗生素是天然产物或其衍生物，通过筛选土壤微生物获得。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。同时，绝大多数细菌，99%的种类，不容易在体外生长，被称为未培养的。我们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，扩散室返回环境。不同生物的分离菌落在培养皿中生长，随后再接种到新的培养皿中产生能够在体外常规培养皿中生长的“驯化”变体。我们的初步研究结果表明，这是一种新型抗菌剂的极好来源。然而，即使有了这种以前无法获得的资源，大部分化学努力仍然浪费在重新发现已知化合物上。我们认为，如果发现集中在一种特定物种的化合物上，这个问题就可以解决。对于结核分枝杆菌，已经发现了几种合成的化合物，它们分别是INH、乙硫酰胺和吡嗪酰胺。迄今为止，对结核分枝杆菌有特异性作用的天然化合物尚未被描述。这意味着筛选特异性抗m。结核病化合物将产生打击，将有很大的可能性是新的物质。然后，一个特定的屏幕将在很大程度上取代费力的重新复制。其基本原理是同时筛查结核分枝杆菌和另一种不同的细菌——金黄色葡萄球菌。初步筛选表明，未培养菌提取物对结核分枝杆菌的特异性命中率为1.5%。该屏幕将在拟议的项目中进行优化。为了正确地验证筛选，我们将需要证明它确实能够识别特异性对抗结核分枝杆菌的新化合物。因此，我们将重复这些打击，确定未知的结构，并建立它们的行动模式。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。一种独特的、未开发的来源——未培养的细菌——与一种特定的筛选相结合，可能会产生对抗耐药结核分枝杆菌的新化合物。

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.