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

描述(申请人提供)：正电子发射断层扫描放射性同位素，如碳-11(11C)和氟-18(18F)，将被加入目前的抗生素，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(S)。将采用保留非共价络合物的分离方法，并在不改变药物结构的情况下合并PET标记。因此，这种方法代表了对现有方法的巨大改进，在现有方法中，药物分子被共价修饰以创建亲和力基质或光亲和标记，并将为合理的药物设计提供关键信息。与依赖于传统放射性标记(14C和~3H)的方法相比，使用PET放射性同位素具有几个优势，例如在灵敏度、蛋白质识别的简便性和体内药物成像能力方面的显著提高。重要的是，将PET标记引入病原体特异性生物分子将有助于使用PET来检测和定位感染患者的病原体。最初的重点将放在结核分枝杆菌和耐甲氧西林金黄色葡萄球菌等病原体上，但这种方法具有普遍的适用性。目的1确定结核一线药物利福平和异烟肼的蛋白质靶点(S)和新型微生物脂肪酸生物合成抑制剂的靶点(S)。将开发合成方法，在不改变每个分子结构的情况下，将11C或18F标记快速整合到药物中。然后，细菌将暴露在标记药物中，并在非变性条件下使用尺寸排除层析和等电聚焦与在线辐射检测等方法进行分级，以识别包含蛋白质-药物复合体的部分。在允许样品腐烂后，将使用标准蛋白质组学方法识别蛋白质药物靶标。在目标2中，在目标1中开发的标记化合物将用于使用正电子发射计算机断层扫描成像药物在健康对照动物体内的组织分布和代谢情况。这一目标将评估不同给药途径对靶器官如肺的药物生物利用度的影响。最终，成像研究将扩展到感染的动物模型和感染患者。与公共卫生相关：持续需要开发对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，由于缺乏复杂的方法来确定药物如何在生命系统中发挥作用，新药的合理开发受到了阻碍。目前这项提议的目标是开发一种可以直接确定药物在细胞内的蛋白质靶标的方法。这种方法依赖于加入短寿命的放射性同位素，这些放射性同位素会衰减以发射穿透人体的光子，因此开发的试剂也应该有助于直接检测感染患者的病原体。

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.