Applications of Mass Spectrometry to Membrane Protein Drug Development

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，包括人类蛋白质靶标，我们有一个前所未有的机会来研究和合理化药物对这些重要靶标的作用。这些重要蛋白质的结构才刚刚出现的原因之一是这些复合体的固有性质，这些性质多年来阻碍了它们的研究；具体地说，它们的低表达水平和较差的溶解性导致了必须克服的实际挑战。由于原子结构和机理的洞察正在出现，因此需要新的方法来评估它们的药物结合特性，并对从其他生物物理方法收集的数据做出贡献。质谱学就是这样一种方法，它提供了对膜蛋白性质的迷人洞察力。没有保护涂层的复杂性，需要溶解，气相可以为研究膜蛋白提供一个理想的环境，从双层中释放出来，但通常保持关键的脂结合。我们热衷于通过将这些观察扩展到脂类和药物的同时作用来进一步发展我们的初步发现，即脂类可以调节膜蛋白的性质。我们选择了三个不同的主题，都对药物开发具有高度的战略重要性，在这里简要介绍。细菌抵抗抗生素挑战的能力是对人类健康的全球性威胁。细菌使用各种机制来做到这一点，包括通过嵌入细胞膜的泵来主动驱逐药物，或者通过控制药物通过毛孔进口，毛孔是由其外层细胞膜中的蛋白质形成的。我们研究计划的一个目标是应用质谱学来获得对这两种机制的新见解。具体地说，我们将研究脂类在保持药物进口的开放毛孔和加快药物通过泵运输出去方面所起的作用。由于许多药物现在以膜蛋白为靶点，通过不加区别地与其他膜蛋白结合而产生了过多的不良副作用。这些包括抗精神病药物与人类葡萄糖转运蛋白的结合，抗艾滋病毒药物与参与衰老的蛋白质的结合，抗癌药物与多药耐药泵的结合，以及与未知功能的人类转运蛋白的结合。因此，第二个目标是开发和应用质谱学方法来研究药物与这些靶点结合的不良副作用。我们研究方案的第三个目标涉及研究保护细菌细胞膜的武器库的组装和合成所涉及的复合体。在这些复合体中，一个折叠并组装毛孔蛋白，另一个协调形成肽聚糖层。因此，这两种药物都是抗生素干预的有吸引力的目标。例如，如果我们能发现阻止毛孔蛋白折叠和组装的新靶点，我们就可以增加它们的合成，以改善药物进口。类似地，如果我们能够控制保护性多肽多糖层的合成，我们可能会影响细菌的生存、它们的分裂和药物的进口。因此，总体而言，这一研究方案将有一些受益者。首先，它将使那些使用质谱学的人受益，因为他们发现了洞察膜蛋白结构生物学的新方法。其次，研究结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目标或纳米技术设备的公司。在对耐药性的长期解释机制中，揭开药物的不良副作用并了解病原菌的细胞壁生物合成将影响全球人类健康。

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.