'Last step' enzymatic [18F]-labelling of peptides for Positron Emission Tomography (PET)

正电子发射断层扫描（PET）是临床上最灵敏的功能成像方法，在西方世界和发展中国家的应用正在迅速增长，特别是作为癌症和退行性神经系统疾病的诊断成像工具。欧洲、美国和亚洲的许多大型医院和临床研究中心现在都在当地调试回旋加速器并开发PET研究设施。氟-18是PET的重要同位素。它的半衰期相对较长（109分钟），很容易以[18F]-氟离子的形式在回旋加速器中以非常高的比活度（GBq）从氧-18水中生成。因此，需要开发用于PET标记的C-[18F]F键形成的新方法，总体而言，氟化学与制药/医疗应用之间的联系很强。自20世纪50年代以来，大约20%的药品含有氟原子，所有新药品的生物分布都需要通过PET来探索，作为临床试验的一部分。此外，新的PET示踪剂作为疾病状态指标的早期诊断工具也是有需求的。新的氟化学需要满足不断增长的需求和动态PET研究社区无论是在英国和国际。本提案旨在开发一种新的方法，将氟化物-18特异性地纳入肽和蛋白质中。在这个建议中，我们的目标是开发一种新的酶，可以从氟离子形成C-F键。氟酶于2002年在圣安德鲁斯被发现（Nature, 2002, 416,279），并被过度表达，其结构（x射线）和机制被阐明。该酶催化氟离子与s -腺苷- l-蛋氨酸（SAM）反应生成5′-FDA和l-蛋氨酸。它已被证明是一种由无机[18F]氟生成C-18F键的化学选择性生物转化方法。然而，我们发现了底物特异性的弱点。我们发现，在一个非常特定的位置，我们可以在底物上附着一个连接体，它将被酶接受，这个连接体提供了一个锚点，使分子线（聚乙二醇）与所选的肽分子相连。所选择的肽是那些识别体内癌细胞的肽，称为归巢肽，或称为“词缀体”的小抗体，用于识别肿瘤细胞。这样我们就可以用酶来连接氟-18同位素。重要的优点是氟-18是在水中产生的，酶在中性ph下在水中起作用。而且肽很好地溶于水，因此标记可以在没有使用有机溶剂的情况下对这些生物分子进行。这呈现出诱人的可能性。氟酶是合成氟-18 PET中使用的酶的唯一例子，在这方面，它提供了一种全新的结合氟的方法。在实际应用中，由于PET使用的是皮摩尔[18F]氟离子，但过表达的氟酶以mg/ml（微米）存在，因此由于酶的大量摩尔过剩，动力学有利于C-18F的合成。这是圣安德鲁斯大学和阿伯丁大学之间的一项研究合作，用于标记肽和蛋白质的酶方法将在圣安德鲁斯开发，放射性标记协议将在位于阿伯丁皇家医院的阿伯丁PET中心进行。该研究的主要重点将集中在中性环境条件下肽的快速标记。这项研究的目的是为不断增长的PET放射化学家国际研究社区建立更广泛的应用新方法，我们有雄心通过与以PET为基础的公司（如Imanova）和我们与格拉斯哥Beatson癌症研究所的既定互动，将这些方法转化为临床。

Positron emission tomography (PET) is the most sensitive functional imaging method clinically and it application is growing rapidly through the Western world and developing countries, particularly as a diagnostic imaging tool for cancers and degenerative neurological disorders. Many major hospitals and clinical research centres in the Europe, the US and Asia are now commissioning cyclotrons and developing PET research facilities locally. Fluorine-18 is an important isotope for PET. It has a relatively long half-life (109 mins) and is readily generated in a cyclotron in the form of [18F]-fluoride ion, in very high specific activity (GBq's) from oxygen-18 water. As a consequence new methods to develop C-[18F]F bond formation for PET labelling are in demand, in general the link between fluorine chemistry and pharmaceutical/medical applications is strong. Approximately 20% of all pharmaceutical, since the 1950s, contain a fluorine atom and the bio-distribution of all new pharmaceutical products are required to be explored by PET, as part of clinical trials. Also new PET tracers are in demand as tools for early diagnosis as indicators of disease states. New fluorine chemistry is required to meet the demands of a growing and dynamic PET research community both in the UK and internationally. This proposal aims to develop a novel methodology for incorporating fluoride-18 specifically into peptides and proteins. In this proposal we aim to exploit a novel enzyme which can form C-F bonds from fluoride ion. The fluorinase enzyme was discovered in 2002 (Nature, 2002, 416, 279) in St Andrews and it has been over-expressed and its structure (X-ray) and mechanism elucidated. The enzyme catalyses the reaction of fluoride ion and S-adenosyl-L-methionine (SAM) to generate 5'-FDA and L-methionine. It has proven to be a chemoselective biotransformation method for generating C-18F bonds from inorganic [18F]-fluoride. However we have found a weakness in the substrate specificity. We find that at a very specific location we can attach a linker to the substrate, and it will be accepted by the enze, this linker provides an anchor point to run a molecular line (poly ethylene glycol) to a peptide molecule of choice. The chosen peptides are those that identify cancer cells in the body, known as homing peptides, or small antibodies called 'affibodies' that identify tumour cells. In this way we can use the enzyme to attach the fluorine-18 isotope. The important advantage is that the fluoride-18 is generated in water, and the enzyme functions in water at neutral pH. Also peptides are nicely soluble in water, so the labelling can take place without the difficulty of using organic solvents for these biomolecules. This presents attractive possibilities. The fluorinase is the only example of an enzyme used in fluorine-18 PET synthesis and in this regard it offers an entirely new method for incorporating fluorine. In practical terms it has emerged to be particularly appropriate, because PET uses picomolar [18F]-fluoride ion, but the over-expressed fluorinase enzyme is present at mg/ml (microM), and therefore the kinetics favour C-18F synthesis due to a large molar excess of enzyme. This is a research collaboration between the Universities of St Andrews and Aberdeen where the enzymatic methods for labelling the petides and proteins will be developed in St Andrews and the radiolabeling protocols carried out at the Aberdeen PET Centre, situated in the Aberdeen Royal Infirmary. The major focus of the research will concentrate on rapid labelling of peptides under neutral ambient conditions.The research in aims to establish new methods for much wider applications by the growing international research community of PET radiochemists and we have ambitions to translate the methods to the clinic through interactions with PET based companies such an Imanova and our established interactions with the Beatson Cancer Institute in Glasgow.