Using PET isotopes to identify drug targets and to diagnose infectious diseases

描述（由申请人提供）：正电子发射断层扫描（PET）放射性同位素，如碳-11 （11C）和氟-18 (18F)，将被纳入目前的抗生素，如结核病药物异烟肼和利福平，以便直接识别活细胞中这些分子的蛋白质靶标。将采用保留非共价复合物的分离方法，并且在不改变药物结构的情况下加入PET标签。因此，这种方法代表了对现有方法的巨大改进，其中药物分子被共价修饰以创建亲和矩阵或光亲和标签，并将为合理的药物设计提供关键信息。与传统的放射性标记（14C和3H）相比，PET放射性同位素的使用有几个优点，例如灵敏度的显著提高，蛋白质鉴定的便利性和体内药物成像的能力。重要的是，将PET标签引入病原体特异性生物分子将有助于使用PET检测和定位感染患者的病原体。最初的重点将是病原体，如结核分枝杆菌和耐甲氧西林金黄色葡萄球菌，但该方法具有普遍适用性。目的1将确定一线结核病药物利福平和异烟肼的蛋白靶点，以及新型微生物脂肪酸生物合成抑制剂的靶点。将开发合成方法，在不改变每个分子整体结构的情况下将11C或18F标签快速纳入药物。然后，细菌将暴露于标记的药物中，并在非变性条件下使用诸如尺寸排除色谱和等电聚焦与在线辐射检测等方法进行分离，以识别含有蛋白质-药物复合物的部分。在让样品腐烂后，将使用标准的蛋白质组学方法鉴定蛋白质药物靶点。在Aim 2中，Aim 1中开发的标记化合物将使用PET对健康对照动物的组织分布和药物代谢谱进行成像。本研究将评估不同给药途径对靶器官（如肺）药物生物利用度的影响。最终，影像学研究将扩展到感染动物模型和感染患者。公共卫生相关性：持续需要开发对诸如耐药结核分枝杆菌和耐甲氧西林金黄色葡萄球菌等病原体有效的新药。然而，由于缺乏确定药物如何在生命系统中起作用的复杂方法，新药的合理开发受到了阻碍。当前提案的目标是开发一种方法，在这种方法中，药物在细胞中的蛋白质靶标可以直接确定。这种方法依赖于短寿命放射性同位素的结合，这些同位素衰变后会释放出穿透身体的光子，因此开发的试剂也可以用于直接检测感染患者体内的病原体。

DESCRIPTION (provided by applicant): Positron emission tomography (PET) radioisotopes, such as carbon-11 (11C) and fluorine-18 (18F), will be incorporated into current antibiotics, such as the tuberculosis drugs isoniazid and rifampicin, in order to directly identify the protein target(s) for these molecules in living cells. Separation methods will be employed that that preserve non-covalent complexes, and PET labels will be incorporated without altering the structure of the drug. This approach thus represents a dramatic improvement on existing methods, in which drug molecules are covalently modified in order to create affinity matrices or photoaffinity labels, and will provide critical information for rational drug design. The use of PET radioisotopes has several advantages compared to methods that relay on conventional radiolabels (14C and 3H), such as dramatic improvements in sensitivity, ease of protein identification and the ability to image drugs in vivo. Importantly, the introduction of PET labels into pathogen-specific biomolecules will facilitate the use of PET to detect and localize pathogens in infected patients. The initial focus will be on pathogens such as Mycobacterium tuberculosis and methicillin-resistant Staphylococcus aureus, however the approach has general applicability. Aim 1 will identify the protein target(s) of the front-line tuberculosis drugs rifampicin and isoniazid as well as the target(s) of novel microbial fatty acid biosynthesis inhibitors. Synthetic methods will be developed to rapidly incorporate 11C or 18F labels into the drugs without altering the overall structure of each molecule. Bacteria will then be exposed to labeled drugs and fractionated under non-denaturing conditions using methods such as size-exclusion chromatography and isoelectric focusing with in-line radiation detection to identify fractions containing protein-drug complexes. After allowing the samples to decay, protein-drug targets will be identified using standard proteomic methods. In Aim 2 the labeled compounds developed in Aim 1 will be used to image the tissue distribution and metabolic profile of the drugs in healthy control animals using PET. This Aim will evaluate the effect of different routes of drug administration on drug bioavailability to target organs such as the lung. Ultimately, the imaging studies will be extended to animal models of infection and infected patients. PUBLIC HEALTH RELEVANCE: There is a continuous need to develop new drugs that are effective against pathogens such as drug- resistant Mycobacterium tuberculosis and methicillin-resistant Staphylococcus aureus. However, the rational development of new drugs is hindered by the lack of sophisticated methods for determining how drugs work ij living systems. The objective of the current proposal is to develop a method in which the protein targets of drugs in cells can be directly determined. This method relies on the incorporation of short-lived radioisotopes that decay to emit body-penetrating photons, and thus the reagents that are developed should also be useful for directly detecting pathogens in infected patients.