High-Throughput Luminescence Assay for Sulfotransferase Activity

磺基转移酶活性的高通量发光测定

• 批准号: BB/T012099/1
• 负责人: Stephen Butler
• 金额: $ 19.14万
• 依托单位: Loughborough University
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FT012099%2F1
• 关键词: Throughput; Luminescence; Assay; Sulfotransferase; Activity

Living things develop and grow, adapt to constant changes in their environment and energy sources, reproduce and resist disease. These things they achieve through communication between organisms and between cells within an organism, often by changing the chemical make-up of molecules. A common route is to add or remove a small charged chemical such as phosphate or sulfate, which act as switches, altering the information that is communicated. This dictates the biological outcome, which may be as varied as cell movement, cell death, and successful (or not) infection. The addition of a negative sulfate group is performed by specific enzymes called sulfotransferases, but we lack the technology to easily measure the activity of these enzymes.Sulfotransferases catalyse the transfer of sulfate to alcohols (-OH) on proteins, large sugars (glycans) and many small molecules, including hormones. There has been focussed research on small selective subsets of these enzymes and on some of their sulfated products. Glycan sulfation in animals plays key roles in development, wound repair and is associated with ageing, neurodegenerative diseases and infection by viruses. Sulfation of proteins on the amino acid tyrosine results in changes in protein-protein interactions related to immune function and virus infectivity. In plants, tyrosine sulfation produces hormones, and can be used by pathogens as part of their infection strategy. Bacterial sulfotransferases can sulfate a wider range of small molecule targets than animals, and some of these enzymes have potential as replacements of currently unsustainable chemical production of personal care and food products.Efforts to understand the global significance of sulfation and to harness it have faced an unsurmountable obstacle: there is no simple and accessible means to measure the sulfation of a target by a sulfotransferase. The consequence is piecemeal research, which is largely unable to deliver the depth and breadth of fundamental knowledge and advances necessary to harness sulfation for our benefit in animal and plant health, and in product synthesis.Our proposal aims to develop a simple, cheap and versatile assay technology that will allow all researchers to measure the addition of a sulfate to any biological or indeed man-made molecule by a sulfotransferase. Such assays have proven to be the key to understanding and exploiting related chemical modifications to biological molecules, such as phosphorylation.Sulfotransferases use a universal sulfate donor, PAPS, which upon transfer of its sulfate group becomes PAP. Measuring PAP provides a direct measure of sulfation. We will develop molecular probes based on a rare earth element, Europium, which emit red light upon bind reversibly to PAP. Measurement of the emitted light is very sensitive and provides a real-time readout of the activity of the sulfotransferase. This will be independent of the molecule the sulfotransferase is attaching the sulfate to. In this way the assay will be fast, real-time and eliminate the need for chemically modified reagents, radioactivity, antibodies and expensive equipment not generally available. Our proposed molecular probes have enormous potential to make the measurement of sulfotransferase activity routine and reduce the cost and time required to conduct high-throughput screening assays. These probes will provide a vital step towards the rapid, accurate determination of sulfotransferase kinetics and mechanism. This will enable better selection and validation of sulfotransferase inhibitors (drug leads), and of mutant enzymes for industrial use. The new technology will pave the way for understanding biological sulfation and exploiting it in the contexts of health and disease, and of product manufacture.

生物发展和生长，适应环境和能源的不断变化，繁殖和抵抗疾病。它们通过生物之间和生物内部细胞之间的交流来实现这些功能，通常是通过改变分子的化学组成。一种常见的方法是添加或移除少量带电的化学物质，如磷酸盐或硫酸盐，它们起到开关的作用，改变所传递的信息。这决定了生物学结果，可能是多种多样的细胞运动、细胞死亡和成功（或不）感染。负硫酸盐基团的加入是由一种叫做硫转移酶的特殊酶来完成的，但我们缺乏容易测量这些酶活性的技术。硫转移酶催化硫酸盐在蛋白质、大糖（聚糖）和许多小分子（包括激素）上转化为醇（-OH）。人们对这些酶的小选择性亚群及其一些硫酸化产物进行了重点研究。动物体内的聚糖磺化在发育、伤口修复、衰老、神经退行性疾病和病毒感染中起着关键作用。氨基酸酪氨酸上蛋白质的硫酸化导致与免疫功能和病毒感染性相关的蛋白质相互作用的变化。在植物中，酪氨酸硫酸化产生激素，并可被病原体用作其感染策略的一部分。与动物相比，细菌硫转移酶可以对更广泛的小分子靶标进行硫酸处理，其中一些酶有可能取代目前个人护理和食品中不可持续的化学生产。了解硫酸化的全球意义和利用它的努力面临着一个无法克服的障碍：没有简单和可获得的方法来测量硫转移酶对目标的硫酸化。其结果是零碎的研究，在很大程度上无法提供深度和广度的基础知识和必要的进展，以利用硫酸化为我们在动植物健康和产品合成方面的利益。我们的建议旨在开发一种简单、廉价和通用的分析技术，使所有研究人员能够通过硫转移酶测量任何生物或人造分子中硫酸盐的添加。这样的分析已被证明是理解和利用生物分子的相关化学修饰的关键，例如磷酸化。硫转移酶使用一种通用的硫酸盐供体，PAPS，在其硫酸盐基团转移后成为PAP。测量PAP可直接测量硫酸化。我们将开发基于稀土元素铕的分子探针，它在与PAP可逆结合时发出红光。发射光的测量是非常敏感的，并提供实时读数的活性的硫转移酶。这与硫转移酶连接硫酸根的分子无关。通过这种方法，分析将是快速、实时的，并且不需要化学修饰试剂、放射性、抗体和昂贵的设备。我们提出的分子探针具有巨大的潜力，可以使硫转移酶活性的测量成为常规，并减少进行高通量筛选试验所需的成本和时间。这些探针将为快速、准确地测定硫转移酶动力学和机制提供重要的一步。这将有助于更好地选择和验证硫转移酶抑制剂（药物先导）和用于工业用途的突变酶。这项新技术将为理解生物硫酸化并在健康和疾病以及产品制造方面加以利用铺平道路。

项目成果

Advances in anion binding and sensing using luminescent lanthanide complexes.

使用发光灯笼型复合物在阴离子结合和传感的进步。

• DOI: 10.1039/d0sc05419d
• 发表时间: 2021-01-26
• 期刊: Chemical science
• 影响因子: 8.4
• 作者: Bodman SE;Butler SJ
• 通讯作者: Butler SJ

Anion binding to a cationic europium(III) probe enables the first real-time assay of heparan sulfotransferase activity.

• DOI: 10.1039/d1ob02071d
• 发表时间: 2022-01-19
• 期刊: Organic & biomolecular chemistry
• 影响因子: 3.2
• 作者: Wheeler S;Breen C;Li Y;Hewitt SH;Robertson E;Yates EA;Barsukov IL;Fernig DG;Butler SJ
• 通讯作者: Butler SJ

Sterically demanding macrocyclic Eu(iii) complexes for selective recognition of phosphate and real-time monitoring of enzymatically generated adenosine monophosphate.

• DOI: 10.1039/d1sc05377a
• 发表时间: 2022-03-24
• 期刊: Chemical science
• 影响因子: 8.4
• 作者: Bodman SE;Breen C;Kirkland S;Wheeler S;Robertson E;Plasser F;Butler SJ
• 通讯作者: Butler SJ

Anion binding to a cationic europium(III) probe enables the first real-time assay of heparan sulfotransferase activity

阴离子与阳离子铕 (III) 探针的结合首次实现了乙酰肝素磺基转移酶活性的实时测定

• DOI: 10.33774/chemrxiv-2021-4ftrg
• 发表时间: 2021
• 作者: Wheeler S