Applications of Mass Spectrometry to Membrane Protein Drug Development

质谱在膜蛋白药物开发中的应用

• 批准号: MR/N020413/1
• 负责人: Carol Robinson
• 金额: $ 173.76万
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FN020413%2F1
• 关键词: Applications; Mass; Spectrometry; Membrane; Protein

Membrane proteins constitute the largest class of current drug targets. With many atomic structures now coming to the fore, including human protein targets, we have an unprecedented opportunity to study and rationalise the action of drugs on these important targets. One of the reasons why structures of these important proteins are only just becoming available is the inherent properties of these complexes that have hindered their study over the years; specifically their low expression levels and poor solubility contribute to the practical challenges that had to be overcome. Now that atomic structures and mechanistic insights are emerging,new methods are therefore required to assess their drug-binding properties and to contribute to data gleaned from other biophysical methods. Mass spectrometry is one such method that is providing fascinating insight into the properties of membrane proteins. Without the complication of protective coatings, required for solubility, the gas phase can be an ideal environment for the study membrane proteins, liberated from the bilayer, but often with critical lipid binding maintained. We are keen to develop further to our initial findings that lipids can modulate the properties of membrane proteins by extending these observations to the role of lipids and drugs simultaneously. We have selected three different themes, all of high strategic importance for drug development, described briefly here. The ability of bacteria to resist the challenge of antibiotics is a global threat to human health. Bacteria use a variety of mechanisms to do this including through pumps embedded in cell membranes to actively expel drugs or by controlling import of drugs through pores, formed by proteins in their outer cell-envelopes. One objective of our research programme is to apply mass spectrometry to gain new insight into these two mechanisms. Specifically, we will study the roles of lipids that play in holding open pores for drug import and in speeding up the transport of drugs out through pumps. Since many drugs now target membrane proteins a plethora of unwanted side effects are occurring through indiscriminate binding to other membrane proteins. These include binding of antipsychotic drugs to the human glucose transporter, anti HIV drugs to proteins involved in ageing and anticancer drugs to multidrug resistance pumps and to a human transporter of unknown function. A second objective is therefore to develop and apply mass spectrometry methods to study the unwanted side effects of drug binding to these targets. A third objective of our research programme involves the study of complexes involved in the assembly and synthesis of the armoury that protects the bacterial cell envelope. Among these complexes, one folds and assembles the pore proteins and the other orchestrates the formation of peptidoglycan layer. Both are therefore attractive targets for antibiotic intervention. For example, if we could uncover new targets that prevent folding and assembly of the pore proteins we could increase their synthesis to improve drug import. Similarly if we could control synthesis of the protective peptidoglycan layer we could affect the survival of bacteria, their division and the import of drugs. Overall therefore this research programme will have a number of beneficiaries. Firstly, it will benefit those using mass spectrometry by uncovering new methods to gain insight into the structural biology of membrane proteins. Secondly, the research outcomes will be of interest to pharmaceutical companies and biotechnology industries, particularly those with a focus on membrane protein as targets in human health or nanotechnology devices. In the longer-term interpreting mechanisms of drug resistance, unravelling the unwanted side-effects of drugs and understanding of the cell-wall biosynthesis of pathogenic bacteria will impact global human health.

膜蛋白是目前最大的一类药物靶点。随着许多原子结构的出现，包括人类蛋白质靶点，我们有了前所未有的机会来研究和合理化药物对这些重要靶点的作用。这些重要蛋白质的结构刚刚变得可用的原因之一是这些复合物的固有性质多年来阻碍了它们的研究;特别是它们的低表达水平和低溶解度导致了必须克服的实际挑战。现在，原子结构和机制的见解正在出现，因此需要新的方法来评估其药物结合特性，并有助于从其他生物物理方法收集的数据。质谱法就是这样一种方法，它提供了对膜蛋白性质的迷人见解。没有复杂的保护涂层，所需的溶解度，气相可以是一个理想的环境，研究膜蛋白，从双层释放，但往往与关键的脂质结合保持。我们热衷于进一步发展我们的初步发现，脂质可以调节膜蛋白的性质，通过扩展这些观察脂质和药物的作用，同时。我们选择了三个不同的主题，都具有高度的战略重要性，药物开发，简要介绍了这里。细菌抵抗抗生素挑战的能力是对人类健康的全球性威胁。细菌使用多种机制来做到这一点，包括通过嵌入细胞膜中的泵来主动排出药物，或者通过控制药物通过由其外部细胞包膜中的蛋白质形成的孔的输入。我们的研究计划的一个目标是应用质谱法来获得这两种机制的新见解。具体来说，我们将研究脂质在保持药物输入的开放孔和加速药物通过泵的运输中所起的作用。由于许多药物现在靶向膜蛋白，因此通过不加选择地结合其他膜蛋白而发生过多的不必要的副作用。这些包括抗精神病药物与人葡萄糖转运蛋白的结合，抗HIV药物与衰老相关蛋白的结合，抗癌药物与多药耐药泵和未知功能的人转运蛋白的结合。因此，第二个目标是开发和应用质谱方法来研究药物与这些靶点结合的不良副作用。我们研究计划的第三个目标涉及研究参与保护细菌细胞被膜的军械库组装和合成的复合物。在这些复合物中，一个折叠和组装孔蛋白，另一个协调肽聚糖层的形成。因此，两者都是抗生素干预的有吸引力的目标。例如，如果我们能够发现阻止孔蛋白折叠和组装的新靶点，我们就可以增加它们的合成，从而改善药物的输入。同样，如果我们能够控制保护性肽聚糖层的合成，我们就可以影响细菌的生存，它们的分裂和药物的输入。因此，总体而言，这项研究方案将有一些受益者。首先，它将通过发现新方法来深入了解膜蛋白的结构生物学而使那些使用质谱的人受益。其次，研究成果将引起制药公司和生物技术行业的兴趣，特别是那些专注于将膜蛋白作为人类健康或纳米技术设备目标的公司。在解释耐药性的长期机制方面，解开药物的不良副作用和了解致病菌的细胞壁生物合成将影响全球人类健康。

项目成果

Native Desorption Electrospray Ionization Liberates Soluble and Membrane Protein Complexes from Surfaces.

• DOI: 10.1002/anie.201704849
• 发表时间: 2017-11-13
• 期刊: Angewandte Chemie (International ed. in English)
• 作者: Ambrose S;Housden NG;Gupta K;Fan J;White P;Yen HY;Marcoux J;Kleanthous C;Hopper JTS;Robinson CV
• 通讯作者: Robinson CV

Pyocin S5 Import into Pseudomonas aeruginosa Reveals a Generic Mode of Bacteriocin Transport

• DOI: 10.1128/mbio.03230-19
• 发表时间: 2020-03-01
• 期刊: MBIO
• 影响因子: 6.4
• 作者: Behrens, Hannah M.;Lowe, Edward D.;Kleanthous, Colin
• 通讯作者: Kleanthous, Colin

A Mass-Spectrometry-Based Approach to Distinguish Annular and Specific Lipid Binding to Membrane Proteins

基于质谱的方法来区分环状和特异性脂质与膜蛋白的结合

• DOI: 10.1002/ange.201914411
• 发表时间: 2020
• 期刊: Angewandte Chemie
• 作者: Bolla J

Native Desorption Electrospray Ionization Liberates Soluble and Membrane Protein Complexes from Surfaces

自然解吸电喷雾电离从表面释放可溶性和膜蛋白复合物

• DOI: 10.1002/ange.201704849
• 发表时间: 2017
• 期刊: Angewandte Chemie
• 作者: Ambrose S