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

用于正电子发射断层扫描 (PET) 的肽的“最后一步”酶促 [18F] 标记

• 批准号: EP/M01262X/1
• 负责人: David O'Hagan
• 金额: $ 43.51万
• 依托单位: University of St Andrews
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=EP%2FM01262X%2F1
• 关键词: Last; step; enzymatic; 18F; labelling

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.

正电子发射断层扫描(PET)是临床上最敏感的功能成像方法，其应用在西方世界和发展中国家迅速发展，特别是作为癌症和退行性神经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正在调试回旋加速器，并在当地发展PET研究设施。氟-18是一种重要的正电子发射体素。它的半衰期相对较长(109分钟)，很容易在回旋加速器中以[18F]-氟离子的形式从氧-18水中产生非常高的比活度(GBq‘s)。因此，开发用于PET标记的C-[18F]F键形成的新方法是需要的，总的来说，氟化学和制药/医疗应用之间的联系是很强的。自20世纪50年代以来，大约20%的药物含有氟原子，作为临床试验的一部分，所有新药物的生物分布都需要通过正电子发射计算机断层扫描(PET)进行探索。此外，人们还需要新的PET示踪剂作为早期诊断的工具，作为疾病状态的指标。为了满足英国和国际上不断增长和充满活力的PET研究社区的需求，需要新的氟化学。这项提议旨在开发一种新的方法，专门将氟-18掺入多肽和蛋白质中。在这项提议中，我们的目标是开发一种新型的酶，它可以从氟离子中形成C-F键。该氟化酶于2002年(Natural，2002,416,279)在圣安德鲁斯细菌中被发现，并已过表达，其结构(X-射线)和作用机制已被阐明。该酶催化氟离子与S-腺苷-L-蛋氨酸反应生成5‘-FDA和L-蛋氨酸。它已被证明是一种从无机[18F]-氟化物生成C-18F键的化学选择性生物转化方法。然而，我们发现了底物专一性的弱点。我们发现，在一个非常特定的位置，我们可以将一个连接物连接到底物上，它将被Enze接受，这个连接物提供了一个锚点，将分子线(聚乙二醇)连接到所选择的多肽分子上。所选择的多肽是那些识别体内癌细胞的多肽，被称为归巢多肽，或者是识别肿瘤细胞的小抗体，称为“亲和体”。通过这种方式，我们可以使用酶来连接氟-18同位素。重要的优点是，氟-18是在水中产生的，这种酶在中性pH的水中发挥作用。此外，多肽在水中很好地溶解，因此可以进行标记，而不需要使用有机溶剂来标记这些生物分子。这带来了诱人的可能性。氟化酶是用于氟-18PET合成的唯一一种酶的例子，在这方面它提供了一种结合氟的全新方法。在实践中，它已经显得特别合适，因为PET使用皮摩尔[18F]-氟离子，但过度表达的氟化酶存在于毫克/毫升(微米)，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，在那里将在圣安德鲁斯开发标记肽段和蛋白质的酶方法，并在位于阿伯丁皇家医院的阿伯丁PET中心进行放射性标记方案。这项研究的主要重点将集中在中性环境条件下的多肽快速标记。该研究旨在为不断增长的国际PET放射化学家研究社区建立新的更广泛的应用方法，我们的雄心是通过与基于PET的公司(如Imanova)的互动以及我们与格拉斯哥Beatson癌症研究所的已建立的互动，将这些方法转化为临床。

项目成果

Exploration of a potential difluoromethyl-nucleoside substrate with the fluorinase enzyme.

用氟化酶探索潜在的二氟甲基核苷底物。

• DOI: 10.1016/j.bioorg.2015.11.003
• 发表时间: 2016
• 期刊: Bioorganic chemistry
• 影响因子: 5.1
• 作者: Thompson S

Enzymatic Fluorination of Biotin and Tetrazine Conjugates for Pretargeting Approaches to Positron Emission Tomography Imaging.

生物素和四嗪缀合物的酶促氟化用于正电子发射断层扫描成像的预靶向方法。

• DOI: 10.1002/cbic.201800234
• 发表时间: 2018
• 期刊: a European journal of chemical biology
• 作者: Lowe PT

Identification of a fluorometabolite from Streptomyces sp. MA37: (2R3S4S)-5-fluoro-2,3,4-trihydroxypentanoic acid.

• DOI: 10.1039/c4sc03540b
• 发表时间: 2015-02-01
• 期刊: Chemical science
• 影响因子: 8.4
• 作者: Ma L;Bartholome A;Tong MH;Qin Z;Yu Y;Shepherd T;Kyeremeh K;Deng H;O'Hagan D
• 通讯作者: O'Hagan D

An enzymatic Finkelstein reaction: fluorinase catalyses direct halogen exchange.

酶促芬克尔斯坦反应：氟化酶催化直接卤素交换。

• DOI: 10.1039/c9ob01625b
• 发表时间: 2019
• 期刊: Organic & biomolecular chemistry
• 影响因子: 3.2
• 作者: Lowe PT

Last-Step Enzymatic [(18) F]-Fluorination of Cysteine-Tethered RGD Peptides Using Modified Barbas Linkers.

• DOI: 10.1002/chem.201601361
• 发表时间: 2016-07
• 期刊: Chemistry
• 作者: Qing-zhi Zhang;S. Dall’Angelo;I. Fleming;L. Schweiger;M. Zanda;D. O'Hagan