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

描述（由申请人提供）：结核病（TB）是一种由结核分枝杆菌引起的细菌性疾病，其具有挑战性的疾病管理以及多重/广泛耐药菌株的日益增多已成为严重的公共卫生问题。虽然这在全球范围内主要影响发展中国家和艾滋病毒/艾滋病问题流行的地区，并导致迫切需要找到新的抗结核先导化合物。通过筛选合成文库来寻找此类化合物的成功率的缺乏将兴趣的焦点重新引导到化学多样性的天然产物上。经典的生物活性导向分离的限制是劳动强度，时间承诺，最重要的是，在检测，分离和表征生物活性的次要成分的难度。目前的探索性项目将为快速检测、分离和鉴定抗结核天然产物的变革性概念奠定基础。该技术将在培养的放线菌菌株的提取物上开发，并将是在一组预选的抗结核活性放线菌菌株上进行大规模抗结核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，该分析环境允许对平面色谱样品分离进行质谱分析，并平行评价其生物活性特征。将质谱与生物活性数据相结合将产生直接将抗TB活性与结构信息相关联的二维图谱。通过在分离过程的早期阶段使用活性成分的化学性质的知识，可以显著缩短分离过程。可以基于基于极性的GUESS原理用逆流色谱法定制特定方案和/或直接升级到制备型TLC。平面色谱法（高效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MS光谱学（LC-MS 2）的最新技术将用于靶向分离先导化合物。已知的化学结构将根据其分子量进行去复制，如从MS（和LC-MS 2）和EI数据库（NIST，Wiley）搜索中收集的。新的或其他重要的分离的活性成分的完整结构解析将利用最先进的基于1D/2D NMR和MS的结构信息，并将得到新方法学的支持，如冷冻探针NMR、基于定量NMR和现代抗TB活性评估的核指纹图谱和纯度-活性关系。TLC-MS-（TB）-生物自显影将与已建立的技术进行比较，优化和验证。该概念通常可应用于天然产物药物发现以处理大量样品，并且在我们的情况下将应用于预先选择的抗结核活性放线菌菌株。我们期望这种新技术的应用将导致发现过多的新的、迄今为止未鉴定的抗TB天然产物，并且这些天然产物中的一种或多种将是用于TB药物开发的可行的先导化合物。

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.