Development of a transformative approach to the rapid detection, isolation, and i

描述(申请人提供)：结核病(TB)的疾病管理具有挑战性，这是一种由结核分枝杆菌引起的细菌疾病，而且越来越多的多重/广泛耐药菌株已成为一个严重的公共卫生问题。虽然是全球性的，但这主要影响到艾滋病毒/艾滋病问题普遍存在的发展中国家和地区，并导致迫切需要寻找新的抗结核病先导化合物。在通过筛选合成文库来寻找此类化合物方面缺乏成功，这使得人们的兴趣焦点回到了化学上多样化的天然产物上。传统的生物活性导向分离的限制是劳动强度、时间投入，最重要的是，检测、分离和表征生物活性次要成分的难度。目前的探索性项目将为快速检测、分离和鉴定抗结核病天然产物的变革性概念奠定基础。这项技术将在培养的放线菌菌株的提取物上开发，并将是在一组预先选择的抗结核活性放线菌菌株上进行大规模抗结核病药物发现项目的先决条件。将开发一种新的TLC-MS-(TB)生物自显影方法，用于在分析环境中直接鉴定活性关键化合物，从而允许对平面色层样品分离进行质谱分析，并同时评估其生物活性。将质谱学与生物活性数据相结合，将生成直接将抗结核活性与结构信息相关联的二维图。通过在分离过程的早期阶段利用有效成分的化学性质的知识，可以显著缩短分离过程。根据基于极性的猜测原理，可以利用逆流层析来定制特定的方案，和/或直接升级到制备TLC。平面层析(高性能TLC、自动“喷雾”采样、梯度洗脱、半自动提取)、微生物学(TB琼脂覆盖发光检测)和质谱学(LC-MS2)中的最先进技术将用于铅化合物的分离。已知的化学结构将根据它们的分子量进行去复制，如从MS(和LC-MS2)和EI数据库(NIST，Wiley)搜索中收集的。新的或其他重要的分离活性成分的完整结构阐明将利用最先进的1D/2D核磁共振和基于MS的结构信息，并将得到诸如冷冻探针核磁共振、核指纹图谱和基于定量核磁共振和现代抗结核活性评估的纯度-活性关系等新方法的支持。TLC-MS-(TB)-生物自显影将与已有的技术进行比较、优化和验证。这一概念通常可以应用于天然产物药物开发，以处理大量样本，并将在我们的案例中应用于预先选择的抗结核活性放线菌菌株。我们预计，这项新技术的应用将导致发现大量迄今为止尚未确定的新的抗结核病天然产品，其中一种或多种将成为结核病药物开发的可行先导化合物。

DESCRIPTION (provided by applicant): The challenging disease management of tuberculosis (TB), a bacterial disease caused by Mycobacterium tuberculosis, and the increasing abundance of multi/extensive drug-resistant strains has precipitated to a severe public health problem. While global this mostly affects developing countries and regions with prevalent HIV/AIDS problems, and leads to an urgent need to find novel anti-TB lead compounds. The lack of success in finding such compounds by screening synthetic libraries directs the focus of interest back to the chemically diverse natural products. The constraints of classical bioactivity guided isolation are labor intensity, time commitment, and most important, the difficulty in detecting, isolating, and characterizing bioactive minor constituents. The present exploratory project will lay the groundwork for a transformative concept to the rapid detection, isolation, and identification of anti-tuberculosis natural products. The technology will be developed on extracts of cultured actinomycete strains and will be a prerequisite for a large-scale anti- TB drug discovery project on a panel of pre-selected anti-TB active actinomycete strains. A novel TLC-MS-(TB)-bioautography method will be developed for the straight-forward identification of active key compounds in an analytical setting that allows the mass spectrometric analysis of planar chromatographic sample separations and in parallel the evaluation of their biological activity profile. Combining the mass spectroscopic with biological activity data will generate two-dimensional maps that directly correlate the anti-TB activity with structural information. The isolation procedure can significantly be shortened by using the knowledge of the chemical properties of the active principle in an early stage of the separation procedure. A specific protocol can be tailored with counter current chromatography based on the polarity based GUESS principle and/or to directly upscale to preparative TLC. State-of-the-art technology in planar chromatography (high-performance TLC, automated "spray-on" sampling, gradient elution, semi-automatic extraction), microbiology (TB agar-overlay with luminescence detection), and MS spectroscopy (LC-MS2) will be used to target the isolation of lead compounds. Known chemical structures will be dereplicated based on their molecular weight, as gleaned from MS (and LC-MS2) and EI database (NIST, Wiley) search. Full structure elucidation of new or otherwise important isolated active principles will utilize state-of-the-art 1D/2D NMR- and MS-based structural information and will be supported by novel methodology like cryoprobe NMR, nuclear fingerprinting and purity-activity relationships based on quantitative NMR and modern anti-TB activity assessment. TLC-MS-(TB)-bioautography will be compared with established technologies, optimized, and validated. The concept can generally be applied in natural product drug discovery to handle large quantities of samples and will in our case be applied to pre-selected anti-TB active actinomycete strains. We expect that application of this novel technology will result in the discovery of a plethora of new, heretofore unidentified anti-TB natural products and that one or more of these will be a viable lead compound for TB drug development.