Protein aptamers to enable structure-based drug discovery - making the undruggable druggable

蛋白质适配子(PAS)仿射体、纳米体和DARPins)是可以识别和结合蛋白质的分子，以帮助可视化和研究它们。它们可以被修饰以结合特定的靶点，并已被证明与抗体在这一应用中一样有效。然而，与抗体相比，PAS也有许多优点，包括更好的稳定性，更好的到达体内一系列组织的能力，以及更简单的生产。PAS在药物发现和结构生物学领域有许多潜在的应用。它们可以用来将靶子捕捉到固体表面上，或者与荧光化学探针结合起来进行药物结合实验。Pas还可以附着在目标上，以改变其性质，并使其更易于通过现有方法进行结构确定。该项目的主要目标是使用被称为DARPins的PAS，通过促进通常不适合用低温EM成像的小目标的可视化来帮助药物发现过程。低温电子显微镜是一种用电子显微镜观察生物标本的方法。许多细胞内的蛋白质由于体积较小，目前无法用低温电子显微镜观察到。DARPins由重复的模块组成，这些模块的数量可以根据应用而改变，以生产小型或大型DARPins。当几个重复的单元捆绑在一起时，它们也具有刚性的螺旋结构。因此，通过蛋白质工程将DARPins连接到较小的蛋白质靶标上，可以通过增加体积和稳定性，使它们易于通过冷冻EM进行成像。这将通过利用对结构生物学和蛋白质-蛋白质相互作用的理解来为生物问题提供技术解决方案。在使用冷冻EM对DARPin支架进行已知目标成像的评估之后，将对具有尚未解决的结构的新型药物目标的成像进行相同的评估。这将包括被认为很难通过其他手段起诉的目标，同样是因为它们的规模小、灵活性或与其他结构方法不兼容。将针对感兴趣的靶点筛选环肽文库，并将生成的高亲和力环肽工程到DARPin支架上。然后，DARPin支架-靶复合体将被用来使用低温EM确定靶的结构。总体目标是在纽卡斯尔大学癌症药物发现中心方案中实施这一使用PAS实现低温EM的方法，以改进对潜在药物靶点的询问，并促进基于结构的药物发现。

Protein aptamers (PAs eg. Affimers, nanobodies and DARPins) are molecules which can recognise and bind to proteins to help visualise and study them. They can be modified to bind specific targets and have been shown to be as effective as antibodies for this application. However PAs also offer many advantages over antibodies, including better stability, improved ability to reach a range of tissues within the body, and simpler production. There are many potential applications for PAs within the fields of drug discovery and structural biology. They could be used to capture targets onto a solid surface or associated with a fluorescent chemical probe to carry out drug binding experiments. PAs could also be attached to a target to alter its properties and make it more amenable structural determination by current methods. The primary aim of this project is to use PAs known as DARPins to aid the drug discovery process by facilitating the visualisation of small targets usually unsuitable for imaging by cryo EM. Cryo EM is a method by which biological specimens are observed using an electron microscope. Many intracellular proteins cannot currently be visualised by cryo EM due to their small size. DARPins consist of repeating modules, the number of which can be altered depending on the application to produce either small or large DARPins. They also have a rigid, helical structure when several repeating units are bound together. Therefore attaching DARPins through protein engineering to smaller protein targets could make them amenable to imaging via cryo EM by adding bulk and stability. This would offer a technological solution to biological problem through exploiting understanding of structural biology and protein-protein interactions. Following evaluation of DARPin scaffolds for imaging of known targets using cryo EM, the same method will be evaluated for imaging of novel drug targets with structures that remain unsolved. This will include targets which would be considered difficult to prosecute by other means, again due to their small size, flexibility or incompatibility with other structural methods. A cyclic peptides library will be screened against targets of interest, with the resultant high affinity cyclic peptides engineered on to the DARPin scaffold. The DARPin scaffold-target complex will then be used to determine the structure of the target using cryo EM. The overall aim is to implement this methodology of using PAs to enable cryo EM within the Newcastle University Center for Cancer drug discovery programme, in order to improve the interrogation of potential drug targets and facilitate structure based drug discovery.